Pyrazol-3-ones That Activate Pro-apoptotic BAX

ABSTRACT

This application features pyrazol-3-one compounds that activate pro-apoptotic BAX. Also featured are methods of using such compounds, e.g., for the treatment or prevention of diseases, disorders, and conditions associated with deregulated apoptosis of cells (e.g., insufficient apoptosis of diseased or damaged cells or essentially the absence of apoptosis of diseased or damaged cells).

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. Utility application Ser. No. 14/350,847,filed Apr. 10, 2014, which is a U.S. National Phase application under 35U.S.C. §371 of International Patent Application No. PCT/US2012/059799,filed Oct. 11, 2012, which claims the benefit of U.S. ProvisionalApplication No. 61/546,022, filed on Oct. 11, 2011, all of which areincorporated by reference herein.

TECHNICAL FIELD

This application features pyrazol-3-one compounds that activate apro-apoptotic function of BAX. Also featured are methods of using suchcompounds, e.g., for the treatment or prevention of diseases, disorders,and conditions associated with deregulated apoptosis of cells (e.g.,diseased or damaged cells; e.g., insufficient apoptosis of diseased ordamaged cells or reduced apoptosis of diseased or damaged cells).Examples of such diseases, disorders, and conditions include, but arenot limited to, those associated with blockade(s) of cell death pathways(e.g., over-expression of anti-apoptotic BCL-2 proteins), e.g.,hyperproliferative diseases, such as cancer.

BACKGROUND

BCL-2 family proteins are key regulators of the mitochondrial apoptoticpathway in health and disease. The BCL-2 family includes bothpro-apoptotic (e.g., BAX) and anti-apoptotic proteins that form acomplex protein interaction network of checks and balances that dictatecell fate (see, e.g., Danial, N. N. & Korsmeyer, S. J. Cell death:critical control points. Cell 116, 205-19 (2004)).

The α-helical BCL-2 homology 3 (BH3) domains of pro-apoptotic members(e.g., BAX) function as death ligands. Pro-apoptotic member BAX is anexecutioner protein of the BCL-2 family that, when activated, undergoesa structural transformation, which converts it from an inactivecytosolic monomer into a lethal mitochondrial pore (see Gavathiotis, E.,Reyna, D. E., Davis, M. L., Bird, G. H. & Walensky, L. D. BH3-triggeredstructural reorganization drives the activation of proapoptotic BAX. MolCell 40, 481-92 (2010)).

Oligomerization of BAX (and its close homologue BAK) within themitochondrial outer membrane enables the release of apoptogenic factorssuch as cytochrome c and smac/diablo that turn on caspases, theenzymatic effectors of apoptosis (see Liu, X., Kim, C. N., Yang, J.,Jemmerson, R. & Wang, X. Induction of apoptotic program in cell-freeextracts: requirement for dATP and cytochrome c. Cell 86, 147-57 (1996);Li, P. et al. Cytochrome c and dATP-dependent formation ofApaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell91, 479-89 (1997); Du, C., Fang, M., Li, Y., Li, L. & Wang, X. Smac, amitochondrial protein that promotes cytochrome c-dependent caspaseactivation by eliminating IAP inhibition. Cell 102, 33-42 (2000); Wei,M. C. et al. Proapoptotic BAX and BAK: a requisite gateway tomitochondrial dysfunction and death. Science 292, 727-30 (2001)). Theexplicit mechanism by which BAX is triggered and how selectpro-apoptotic BCL-2 proteins directly engage and activate BAX have beenkey questions in the apoptosis field (see, e.g., Youle, R. J. &Strasser, A. The BCL-2 protein family: opposing activities that mediatecell death. Nat Rev Mol Cell Biol 9, 47-59 (2008)).

The α-helical BCL-2 homology 3 (BH3) domains of activated pro-apoptoticmembers (e.g., BAX) can, however, be intercepted and sequestered bystructurally-defined surface grooves within the anti-apoptotic members(see, e.g., Sattler, M. et al. Structure of Bcl-xL-Bak peptide complex:recognition between regulators of apoptosis. Science 275, 983-6 (1997)).The relative levels of death-activating (pro-apoptotic) BH3 domains andanti-apoptotic BH3-binding pockets dictate the cellular response tostress. Cancer cells hijack the survival circuitry of the BCL-2 familypathway, exploiting pathologic overexpression of anti-apoptotic proteinsto stymie physiologic and pharmacologic pro-apoptotic stimuli. Byoverexpressing these anti-apoptotic proteins, cancer cells maintain asurvival advantage in the face of pro-apoptotic stimuli. Thus, theover-expression of anti-apoptotic members is believed to contribute tocancer pathogenesis.

Whereas the mainstay of developmental BCL-2 family therapeutics hasfocused on the loss-of-function strategy of inhibiting anti-apoptoticproteins, direct activation of BAX by select pro-apoptotic BCL-2 membersthat only contain a conserved BH3 domain (“BH3-only” proteins) has alsoemerged as a physiologically relevant mechanism for inducingmitochondrial apoptosis during development and homeostasis (see Ren, D.et al. BID, BIM, and PUMA are essential for activation of the BAX- andBAK-dependent cell death program. Science 330, 1390-3 (2010)).

SUMMARY I

This application features pyrazol-3-one compounds that activate apro-apoptotic function of BAX, making them therapeutically useful fortreating (e.g., controlling, relieving, ameliorating, alleviating, orslowing the progression of) or preventing (e.g., delaying the onset ofor reducing the risk of developing) diseases, disorders, and conditionsassociated with deregulated apoptosis of cells (e.g., diseased ordamaged cells; e.g., insufficient apoptosis of diseased or damagedcells; or lack of apoptosis of diseased or damaged cells). Examples ofsuch diseases, disorders, and conditions include (but are not limitedto) those associated with blockade(s) of cell death pathways (e.g.,over-expression of anti-apoptotic BCL-2 proteins), e.g.,hyperproliferative diseases, such as cancer (e.g., leukemia, e.g., acutelymphoblastic leukemia (“ALL”) or acute myelogenous leukemia (“AML”);e.g., chronic lymphoblastic leukemia (“CLL”) or chronic myelogenousleukemia (“CML”)). While not wishing to be bound by theory, it isbelieved that the compounds described herein induce and increaseapoptosis in target cells (e.g., pathogenic cells including, but notlimited to, cancer cells), thereby suppressing tumor growth and/orproliferation. It is further believed that increasing apoptosis in suchtarget cells reestablishes the normal apoptotic control that, duringhomeostasis, is associated with a regulated balance between pro- andanti-apoptotic protein functions.

[A] In some embodiments, the compounds described herein directlyactivate BAX by direct binding to BAX.

In some embodiments, the compounds described herein selectively bind toand activate BAX. For example, the compounds described hereinselectively bind to and activate BAX in the presence of one (or more)different BCL-2 proteins, e.g., in the presence of one (or more) otherpro-apoptotic BCL-2 proteins (e.g., BAK) and/or in the presence of one(or more) anti-apoptotic BCL-2 proteins.

In some embodiments, the compounds described herein directly activateBAX by direct binding to BAX; and selectively bind to and activate BAX,e.g. selectively bind to and activate BAX in the presence of one (ormore) different BCL-2 proteins, e.g., in the presence of one (or more)other pro-apoptotic BCL-2 proteins (e.g., BAK) and/or in the presence ofone (or more) anti-apoptotic BCL-2 proteins.

[B] It has been discovered that BAX contains a geographically distinctBH3 binding groove, which has been shown to mediate the directactivation of BAX. Specifically, structural analysis of a BIM BH3 deathdomain in complex with pro-apoptotic BAX uncovered a BH3 interactionsite that, when engaged, results in the direct activation of BAX (seeGavathiotis, E. et al. BAX activation is initiated at a novelinteraction site. Nature 455, 1076-81 (2008)). A BIM BH3 α-helix,structurally reinforced by hydrocarbon stapling, engages BAX at theopposite side of the protein from the canonical BH3-binding groove ofanti-apoptotic proteins (see Gavathiotis, E. et al. BAX activation isinitiated at a novel interaction site. Nature 455, 1076-81 (2008)). SeeFIG. 1A. This BH3 trigger site on BAX is formed by the confluence ofα-helices 1 and 6, and is structurally defined by a hydrophobic groovecomprised of amino acids M20, A24, L27, I31, I133, M137, and L141, and aperimeter of charged and hydrophilic residues, including K21, Q28, Q32,E131, and R134. See FIG. 1B. The flexible loop between α-helices 1 and 2partially overlies the binding site and its displacement by BIM BH3 hasbeen implicated as the first ligand-induced conformational change of theBAX activation mechanism (see Gavathiotis, E., Reyna, D. E., Davis, M.L., Bird, G. H. & Walensky, L. D. BH3-triggered structuralreorganization drives the activation of proapoptotic BAX. Mol Cell 40,481-92 (2010)). For ease of exposition, this activating binding groovethat is discussed at the start of section [III] is sometimes referred toherein as the “BAX trigger site”

In some embodiments, the compounds described herein directly activateBAX by binding to BAX at the BAX trigger site.

In some embodiments, the compounds described herein selectively activateBAX by binding to BAX at the BAX trigger site; e.g., selectivelyactivate BAX in the presence of one (or more) different BCL-2 proteins,e.g., in the presence of one (or more) other pro-apoptotic BCL-2proteins (e.g., BAK) and/or in the presence of one (or more)anti-apoptotic BCL-2 proteins.

[C] In some embodiments, the compounds described herein induce oractivate BAX-dependent or mediated apoptosis (cell death).

[D] In some embodiments, the methods described herein can include invitro methods, e.g., contacting a sample containing BAX (e.g., a cell ortissue containing BAX) with a compound of formula (I) or apharmaceutically acceptable salt thereof (e.g., including any subgeneraor specific compound thereof of formula (I), e.g., formula (I-A))

In some embodiments, the methods described herein can includeadministering a compound of formula (I) or a pharmaceutically acceptablesalt thereof (e.g., including any subgenera or specific compound thereofof formula (I), e.g., formula (I-A)) to a subject (e.g., a subject inneed thereof, e.g., a mammal, such as a human).

[E] Accordingly, in one aspect, methods for activating (e.g., directly,selectively, directly and selectively as defined anywhere herein) BAXand/or inducing or activating BAX-dependent apoptosis are featured,which include contacting BAX with a compound of formula (I) or apharmaceutically acceptable salt thereof:

wherein:

A is N or CH;

X is heteroaryl, which contains 5 ring atoms, wherein from 1-2 of thering atoms is/are independently selected from N, NH, N(C₁-C₃ alkyl), O,and S; wherein:

-   -   X is connected to the pyrazolone nitrogen via a ring carbon atom        in X; and    -   X is optionally further substituted with 1 R^(a):

or

X is phenyl optionally substituted with from 1-5 R^(a);

or

X is heteroaryl, which contains from 8-10 ring atoms, wherein from 1-4of the ring atoms is/are independently selected from N, NH, N(C₁-C₃alkyl), O, and S; wherein:

-   -   X is connected to the pyrazolone nitrogen via a ring carbon atom        in X; and    -   X is optionally further substituted with 1 R^(a):

Y is:

(i) C₆-C₁₀ aryl, which is optionally substituted with from 1-5independently selected R^(b); or

(ii) heteroaryl, which contains from 5-10 ring atoms, wherein from 1-4of the ring atoms is independently selected from N, NH, N(C₁-C₃ alkyl),O, and S; and wherein said heteroaryl ring is optionally substitutedwith from 1-3 independently selected R^(b); or

(iii) C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkoxy, C₁-C₆ thioalkoxy,C₁-C₆ haloalkoxy, or C₁-C₆ halothioalkoxy, each of which is optionallysubstituted with —OH, —NH₂, or —SH;

R¹ is:

(i) C₆-C₁₀ aryl, which is optionally substituted with from 1-5independently selected R^(c); or

(ii) heteroaryl, which contains from 5-10 ring atoms, wherein from 1-4of the ring atoms is independently selected from N, NH, N(C₁-C₃ alkyl),O, and S; and wherein said heteroaryl ring is optionally substitutedwith from 1-3 independently selected Re; or

(iii) —C(O)—(C₆-C₁₀ aryl or heteroaryl, which contains from 5-10 ringatoms as defined in R¹ definition (i) and (ii), respectively, above); or

(iv) hydrogen;

each of R² and R^(a) is, independently, selected from any one of thesubstituents delineated collectively in (a), (b), (c), (d), and (e)below:

(a) C₁-C₈ alkyl or C₁-C₈ haloalkyl, each of which is optionallysubstituted with from 1-2 R^(d);

(b) phenyl that is optionally substituted with from 1-4 R^(e);

(c) heteroaryl containing from 5-6 ring atoms, wherein from 1-4 of thering atoms is independently selected from N, NH, N(C₁-C₃ alkyl),NC(O)(C₁-C₆ alkyl), O, and S; and wherein said heteroaryl is optionallysubstituted with from 1-3 R^(e);

(d) C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl, each of which is optionallysubstituted with from 1-4 independently selected C₁-C₄ alkyl groups; or

(e) —NHC(O)(C₁-C₆ alkyl), —C(O)(C₁-C₆ alkyl); or —C(O)O(C₁-C₆ alkyl);

R^(b), at each occurrence, is independently selected from any one thesubstituents delineated collectively in (aa), (bb) and (cc) below:

(aa) C₁-C₆ alkoxy; C₁-C₆ haloalkoxy; C₁-C₆ thioalkoxy; C₁-C₆thiohaloalkoxy; C₁-C₆ alkyl, C₁-C₆ haloalkyl, —NH(C₁-C₆ alkyl), N(C₁-C₆alkyl)₂, or —NHC(O)(C₁-C₆ alkyl), each of which is optionallysubstituted with —OH, —NH₂, azido (—N₃), or —SH;

(bb) halo; —OH; —CN; nitro; —NH₂; azido; C₂-C₄ alkenyl; C₂-C₄ alkynyl;—C(O)H; —C(O)(C₁-C₆ alkyl); C(O)OH; —C(O)O(C₁-C₆ alkyl); —OC(O)(C₁-C₆alkyl); —SO₂(C₁-C₆ alkyl); —SO₂(C₁-C₆ haloalkyl); —C(O)NH₂;—C(O)NH(C₁-C₆ alkyl); C(O)N(C₁-C₆ alkyl)₂; —SO₂(C₁-C₆ alkyl); —SO₂NH₂;—SO₂NH(C₁-C₆ alkyl); —SO₂N(C₁-C₆ alkyl)₂; —NHCO(C₁-C₆ alkyl), or—NHSO₂(C₁-C₆ alkyl); and

(cc) C₃-C₆ cycloalkyl, C₃-C₆ cycloalkoxy, or heterocyclyl containingfrom 5-6 ring atoms, wherein from 1-2 of the ring atoms of theheterocyclyl is independently selected from N, NH, N(C₁-C₆ alkyl),NC(O)(C₁-C₆ alkyl), O, and S; and each of said ring systems isoptionally substituted with from 1-3 independently selected C₁-C₄ alkylgroups;

each occurrence of R^(c) and R^(e) is, independently, selected from anyone the substituents delineated collectively in (aaa), (bbb), (ccc), and(ddd) below:

(aaa) C₁-C₆ alkoxy; C₁-C₆ haloalkoxy; C₁-C₆ thioalkoxy; C₁-C₆thiohaloalkoxy; C₁-C₆ alkyl, C₁-C₆ haloalkyl, —NH(C₁-C₆ alkyl), N(C₁-C₆alkyl)₂, or —NHC(O)(C₁-C₆ alkyl), each of which is optionallysubstituted with —OH, —NH₂, or —SH; (and optionally benzyloxy);

(bbb) halo; —OH; —CN; nitro; —NH₂; azido; C₂-C₄ alkenyl; C₂-C₄ alkynyl;—C(O)H; —C(O)(C₁-C₆ alkyl); C(O)OH; —C(O)O(C₁-C₆ alkyl); —OC(O)(C₁-C₆alkyl); —SO₂(C₁-C₆ alkyl); —SO₂(C₁-C₆ haloalkyl); —C(O)NH₂;—C(O)NH(C₁-C₆ alkyl); C(O)N(C₁-C₆ alkyl)₂; —SO₂(C₁-C₆ alkyl); —SO₂NH₂;—SO₂NH(C₁-C₆ alkyl); —SO₂N(C₁-C₆ alkyl)₂; —NHCO(C₁-C₆ alkyl),—NHSO₂(C₁-C₆ alkyl); —C(O)O—(CH₂)_(1-3(e.g.,1))—C(O)-(phenyl optionallysubstituted as defined in (ddd) below (e.g., —C(O)O—CH₂—C(O)-(phenyl);

(ccc) L-C₃-C₈ cycloalkyl, C₃-C₆ cycloalkoxy, or L-heterocyclylcontaining from 5-7 ring atoms, wherein from 1-2 of the ring atoms ofthe heterocyclyl is independently selected from N, NH, N(C₁-C₆ alkyl),NC(O)(C₁-C₆ alkyl), NC(O)O(C₁-C₆ alkyl), O, and S; and each of said ringsystems is optionally substituted with from 1-3 independently selectedC₁-C₄ alkyl groups; and wherein L is a bond or C₁-C₆ alkylene; and

(ddd) phenyl, —O-(phenyl), or heteroaryl containing from 5-6 ring atoms,wherein from 1-2 of the ring atoms of the heteroaryl is independentlyselected from N, NH, N(C₁-C₃ alkyl), O, and S; wherein each of saidphenyl and heteroaryl is optionally substituted with from 1-3substituents independently selected from halo; hydroxyl; cyano;—C(O)(C₁-C₆ alkyl); C(O)OH; —C(O)O(C₁-C₆ alkyl); nitro; —NH₂; —NH(C₁-C₆alkyl), N(C₁-C₆ alkyl)₂, —NHC(O)(C₁-C₆ alkyl), C₁-C₆ alkoxy; C₁-C₆haloalkoxy; C₁-C₆ thioalkoxy; C₁-C₆ thiohaloalkoxy; C₁-C₆ alkyl, andC₁-C₆ haloalkyl, wherein said alkyl or alkyl portion is optionallysubstituted with —OH, —NH₂, or —SH; and

R^(d) at each occurrence is, independently, selected from hydroxyl,C₁-C₆ alkoxy; C₁-C₆ thioalkoxy; C₁-C₆ haloalkoxy; C₁-C₆ thiohaloalkoxy;—NH₂; —NH(C₁-C₆ alkyl); N(C₁-C₆ alkyl)₂; —NHC(O)(C₁-C₆ alkyl); cyano;—C(O)H; —C(O)(C₁-C₆ alkyl); —C(O)(C₁-C₆ haloalkyl); C(O)OH; —C(O)O(C₁-C₆alkyl); —C(O)NH₂; —C(O)NH(C₁-C₆ alkyl); C(O)N(C₁-C₆ alkyl)₂; —SO₂(C₁-C₆alkyl); —SO₂NH₂; —SO₂NH(C₁-C₆ alkyl); and —SO₂N(C₁-C₆ alkyl)₂.

One or more of the following can apply.

In some embodiments, X is not phenyl optionally substituted with from1-5 R^(a).

In some embodiments, IV is not substituted directly or indirectly withone or more hydroxyl (—OH) groups.

In some embodiments, X is not phenyl optionally substituted with from1-5 R^(a); and R¹ is not substituted directly or indirectly with one ormore hydroxyl (—OH) groups.

In some embodiments, when R¹ is 2-methoxyphenyl, and R² is C₁-C₈ alkyl(e.g., CH₃), then Y cannot be substituted phenyl, e.g., monosubstitutedphenyl, e.g., phenyl monosubstituted at the para position, e.g.,4-chlorophenyl).

In some embodiments, when R¹ is 2-carboxyphenyl, and R² is C₁-C₈ alkyl(e.g., CH₃), then Y cannot be substituted phenyl, e.g., monosubstitutedphenyl, e.g., phenyl monosubstituted at the para position, e.g.,4-methoxyphenyl).

In some embodiments, R¹ is other than 3-nitro-4-chlorophenyl. In certainembodiments, R¹ is other than 3-nitro-4-chlorophenyl when R² and Y areboth unsubstituted phenyl.

In some embodiments, the compound is other than the compound sometimesreferred to herein as “BAM7.”

In another aspect, compounds having formula (I-A), or a pharmaceuticallyacceptable salt thereof, are featured:

In some embodiments of formula (I-A):

X′ is S;

X′″ is unsubstituted phenyl,

X″ is H or C₁-C₄ alkyl;

R² is:

-   -   C₁-C₄ alkyl; or    -   phenyl that is optionally substituted with from 1-4 R^(e); or or    -   heteroaryl containing from 5-6 ring atoms, wherein from 1-4 of        the ring atoms is independently selected from N, NH, N(C₁-C₃        alkyl), NC(O)(C₁-C₆ alkyl), O, and S; and wherein said        heteroaryl is optionally substituted with from 1-3 R^(e);

R¹² is:

-   -   —C(O)OH;    -   C₂-C₆ alkoxy that is optionally substituted with —NH₂; or    -   heterocyclyl containing from 5-7 ring atoms, wherein from 1-2 of        the ring atoms of the heterocyclyl is independently selected        from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), NC(O)O(C₁-C₆        alkyl), O, and S; and each of which is optionally substituted        with from 1-3 independently selected C₁-C₄ alkyl groups;

each of R¹³ and R¹⁴ is H; and

each occurrence of R^(e) is, independently, halo; cyano; —C(O)(C₁-C₆alkyl); C(O)OH; —C(O)O(C₁-C₆ alkyl); nitro; —NH₂; —NH(C₁-C₆ alkyl),N(C₁-C₆ alkyl)₂, —NHC(O)(C₁-C₆ alkyl), C₁-C₆ alkoxy; C₁-C₆ haloalkoxy;C₁-C₆ thioalkoxy; C₁-C₆ thiohaloalkoxy; C₁-C₆ alkyl, C₃-C₆ cycloalkyl;and C₁-C₆ haloalkyl.

One or more of the following can apply.

In certain embodiments, it is provided that R¹² cannot be —C(O)OH whenR² is unsubstituted phenyl.

In certain embodiments, it is provided that R¹² cannot be —OCH₂CH₃ whenR² is unsubstituted phenyl.

In certain embodiments, it is provided that R¹² cannot be —OCH₂CH₃ whenR² is CH₃.

In other embodiments of formula (I-A):

X′ is NH;

X′″ is unsubstituted phenyl,

X″ is H or C₁-C₄ alkyl;

R² is:

-   -   C₁-C₄ alkyl; or    -   phenyl that is optionally substituted with from 1-4 R^(e); or or    -   heteroaryl containing from 5-6 ring atoms, wherein from 1-4 of        the ring atoms is independently selected from N, NH, N(C₁-C₃        alkyl), NC(O)(C₁-C₆ alkyl), O, and S; and wherein said        heteroaryl is optionally substituted with from 1-3 R^(e);

R¹² is:

-   -   —C(O)OH;    -   C₂-C₆ alkoxy that is optionally substituted with —NH₂; or    -   heterocyclyl containing from 5-7 ring atoms, wherein from 1-2 of        the ring atoms of the heterocyclyl is independently selected        from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), NC(O)O(C₁-C₆        alkyl), O, and S; and each of which is optionally substituted        with from 1-3 independently selected C₁-C₄ alkyl groups;

each of R¹³ and R¹⁴ is H; and

each occurrence of R^(e) is, independently, halo; cyano; —C(O)(C₁-C₆alkyl); C(O)OH; —C(O)O(C₁-C₆ alkyl); nitro; —NH₂; —NH(C₁-C₆ alkyl),N(C₁-C₆ alkyl)₂, —NHC(O)(C₁-C₆ alkyl), C₁-C₆ alkoxy; C₁-C₆ haloalkoxy;C₁-C₆ thioalkoxy; C₁-C₆ thiohaloalkoxy; C₁-C₆ alkyl, C₃-C₆ cycloalkyl;and C₁-C₆ haloalkyl.

[F] In one aspect, methods for treating (e.g., controlling, relieving,ameliorating, alleviating, or slowing the progression of) or preventing(e.g., delaying the onset of or reducing the risk of developing)diseases, disorders, and conditions associated with deregulatedapoptosis of cells (e.g., diseased or damaged cells; e.g., insufficientapoptosis of diseased or damaged cells; or the lack of apoptosis ofdiseased or damaged cells) in a subject in need thereof are featured.The methods include administering to the subject (e.g., an effectiveamount of) a compound of formula (I), or a pharmaceutically acceptablesalt thereof (e.g., including any subgenera or specific compound thereofof formula (I), e.g., formula (I-A)).

[G] In another aspect, methods for treating (e.g., controlling,relieving, ameliorating, alleviating, or slowing the progression of) orpreventing (e.g., delaying the onset of or reducing the risk ofdeveloping) diseases, disorders, and conditions associated withblockade(s) of cell death pathways (e.g., over-expression ofanti-apoptotic proteins BCL-2 proteins) in a subject in need thereof arefeatured. The methods include administering to the subject (e.g., aneffective amount of) a compound of formula (I), or a pharmaceuticallyacceptable salt thereof (e.g., including any subgenera or specificcompound thereof of formula (I), e.g., formula (I-A)).

[H] In a further aspect, methods for treating (e.g., controlling,relieving, ameliorating, alleviating, or slowing the progression of) orpreventing (e.g., delaying the onset of or reducing the risk ofdeveloping) a hyperproliferative disease in a subject in need thereofare featured. The methods include administering to the subject (e.g., aneffective amount of) a compound of formula (I), or a pharmaceuticallyacceptable salt thereof (e.g., including any subgenera or specificcompound thereof of formula (I), e.g., formula (I-A)).

In an aspect, methods for treating (e.g., controlling, relieving,ameliorating, alleviating, or slowing the progression of) ahyperproliferative disease in a subject in need thereof are featured.The methods include administering to the subject (e.g., an effectiveamount of) a compound of formula (I), or a pharmaceutically acceptablesalt thereof (e.g., including any subgenera or specific compound thereofof formula (I), e.g., formula (I-A)).

[I] In still another aspect, methods for treating (e.g., controlling,relieving, ameliorating, alleviating, or slowing the progression of) orpreventing (e.g., delaying the onset of or reducing the risk ofdeveloping) cancer (e.g., leukemia, e.g., ALL or AML; e.g., CLL or CML)in a subject in need thereof are featured. The methods includeadministering to the subject (e.g., an effective amount of) a compoundof formula (I), or a pharmaceutically acceptable salt thereof (e.g.,including any subgenera or specific compound thereof of formula (I),e.g., formula (I-A)).

In an aspect, methods for treating (e.g., controlling, relieving,ameliorating, alleviating, or slowing the progression of) cancer (e.g.,leukemia, e.g., ALL or AML) in a subject in need thereof are featured.The methods include administering to the subject (e.g., an effectiveamount of) a compound of formula (I), or a pharmaceutically acceptablesalt thereof (e.g., including any subgenera or specific compound thereofof formula (I), e.g., formula (I-A)).

[J] In yet another aspect, methods of modulating (e.g., increasing)apoptosis in vitro or in vivo are featured. Also featured are methods ofmodulating (e.g., decreasing) cell division in vitro or in vivo arefeatured. The methods can include contacting a sample containing BAX(e.g., a cell or tissue containing BAX) with a compound of formula (I)or a pharmaceutically acceptable salt thereof (e.g., including anysubgenera or specific compound thereof of formula (I)); or administeringa compound of formula (I) or a pharmaceutically acceptable salt thereof(e.g., including any subgenera or specific compound thereof of formula(I), e.g., formula (I-A)) to a subject (e.g., a subject in need thereof,e.g., a mammal, such as a human).

[K] In some embodiments, the methods described above and throughout thisdisclosure can include one or more of the following features.

The cancer can include carcinomas (originating in the outer layer ofcells of the skin and internal membranes, e.g., breasts, lungs,intestines, skin, prostate, etc.); sarcomas (arising from connectivetissue such as bone, muscle, cartilage and blood vessels), andhematologic malignancies (e.g., lymphomas and leukemias, which arise inthe blood or blood-forming organs such as the spleen, lymph nodes andbone marrow, e.g., leukemias, e.g., ALL or AML; e.g., CLL or CML).Cancer cells can include, for example, tumor cells, neoplastic cells,malignant cells, metastatic cells, and hyperplastic cells.

Non-limiting examples of cancers include breast cancer, prostate cancer,lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma,malignant melanoma, ovarian cancer, brain cancer, primary braincarcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladdercancer, non-small cell lung cancer, head or neck carcinoma, breastcarcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma,Wilms' tumor, cervical carcinoma, testicular carcinoma, bladdercarcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma,prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma,esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma,renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma,malignant pancreatic insulinoma, malignant carcinoid carcinoma,choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervicalhyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocyticleukemia, chronic granulocytic leukemia, acute granulocytic leukemia,acute myelogenous leukemia, chronic myelogenous leukemia, hairy cellleukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma,polycythemia vera, essential thrombocytosis, Hodgkin's disease,non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primarymacroglobulinemia, and retinoblastoma.

In some embodiments, the patient has not been treated with an agent thatcauses a thrombocytopenia-associated condition. In some embodiments thepatient is not suffering from and/or is not a risk from developing athrombocytopenia-associated condition.

In some embodiments, the methods further include administering one ormore additional therapeutic agents (e.g., anticancer/chemotherapeuticagents) and/or techniques (e.g., radiation therapies, surgicalinterventions, and the like) to a subject or in vitro cells, tissues,and organs.

In certain embodiments, the methods further include administering one ormore additional therapeutic agents such as: agents that induceapoptosis; polynucleotides (e.g., anti-sense, ribozymes, siRNA);polypeptides (e.g., enzymes and antibodies); biological mimetics (BH3mimetics); agents that bind to and inhibit anti-apoptotic proteins(e.g., agents that inhibit anti-apoptotic BCL-2 proteins); alkaloids;alkylating agents; antitumor antibiotics; antimetabolites; hormones;platinum compounds; monoclonal or polyclonal antibodies (e.g.,antibodies conjugated with anticancer drugs, toxins, defensins, etc.),toxins, radionuclides; biological response modifiers (e.g., interferons(e.g., IFN-.alpha., etc.) and interleukins (e.g., IL-2, etc.), etc.);adoptive immunotherapy agents; hematopoietic growth factors; agents thatinduce tumor cell differentiation (e.g., all-trans-retinoic acid, etc.);gene therapy reagents (e.g., antisense therapy reagents andnucleotides); tumor vaccines; angiogenesis inhibitors; proteosomeinhibitors: NF kappa .beta. modulators; anti-CDK compounds; HDACinhibitors; and the like.

In certain embodiments, the methods further include administering one ormore additional therapeutic agents that bind to and inhibitanti-apoptotic proteins (e.g., agents that inhibit anti-apoptotic BCL-2proteins), such as ABT-263, obatoclax, gossypol derivatives, IAPinhibitors, and stapled peptides that target anti-apoptotic proteins(MCL-1 SAHB (see, Stewart et al, Nature Chem Biol, 2010), BID SAHB(Walensky et al Science 2004), BAD SAHB (Danial et al Nature Medicine2008), BIM SAHB (Gavathiotis et al Nature 2008), etc.).

In certain embodiments, the methods further include administering one ormore additional therapeutic agents that induce or stimulate apoptosis.Agents that induce apoptosis include, but are not limited to, radiation(e.g., X-rays, gamma rays, UV); kinase inhibitors (e.g., EpidermalGrowth Factor Receptor (EGFR) kinase inhibitor, Vascular Growth FactorReceptor (VGFR) kinase inhibitor, Fibroblast Growth Factor Receptor(FGFR) kinase inhibitor, Platelet-derived Growth Factor Receptor (PDGFR)kinase inhibitor, and Bcr-Abl kinase inhibitors such as GLEEVEC);antisense molecules; antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN, andAVASTIN); anti-estrogens (e.g., raloxifene and tamoxifen);anti-androgens (e.g., flutamide, bicalutamide, finasteride,aminoglutethamide, ketoconazole, and corticosteroids); cyclooxygenase 2(COX-2) inhibitors (e.g., celecoxib, meloxicam, NS-398, andnon-steroidal anti-inflammatory drugs (NSAIDs)); anti-inflammatory drugs(e.g., butazolidin, DECADRON, DELTASONE, dexamethasone, dexamethasoneintensol, DEXONE, HEXADROL, hydroxychloroquine, METICORTEN, ORADEXON,ORASONE, oxyphenbutazone, PEDIAPRED, phenylbutazone, PLAQUENIL,prednisolone, prednisone, PRELONE, and TANDEARIL); and cancerchemotherapeutic drugs (e.g., irinotecan (CAMPTOSAR), CPT-11,fludarabine (FLUDARA), dacarbazine (DTIC), dexamethasone, mitoxantrone,MYLOTARG, VP-16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin,gemcitabine, bortezomib, gefitinib, bevacizumab, TAXOTERE or TAXOL);cellular signaling molecules; ceramides and cytokines; andstaurosporine, and the like.

In some embodiments, the subject can be a subject in need thereof (e.g.,a subject identified as being in need of such treatment, such as asubject having, or at risk of having, one or more of the diseases orconditions described herein). Identifying a subject in need of suchtreatment can be in the judgment of a subject or a health careprofessional and can be subjective (e.g. opinion) or objective (e.g.measurable by a test or diagnostic method). In some embodiments, thesubject can be a mammal. In certain embodiments, the subject can be ahuman.

In certain embodiments, the subject has not previously undergonechemotherapy. In certain embodiments, the subject is not suffering from,or at risk of, thrombocytopenia, such as thrombocytopenia resulting fromchemotherapy, radiation therapy, or bone marrow transplantation astreatment for cancer or lymphoma.

[L] In another aspect, methods of screening for (thereby identifying)compounds that activate BAX are featured. Said methods generally includescreening a compound of formula (I) or a pharmaceutically acceptablesalt thereof (e.g., including any subgenera or specific compound thereofof formula (I), e.g., formula (I-A)) and a test compound. The methodsinclude providing: a compound of formula (I) or a pharmaceuticallyacceptable salt thereof (e.g., including any subgenera or specificcompound thereof of formula (I)); a test compound; a first group ofcells; and contacting the first group of cells with the formula (I)compound and the test compound; and observing the effects of contactingthe first group of cells with the formula (I) compound and the testcompound. In some of these embodiments, the methods further provide theadditional step of comparing the effects observed in the first cellsagainst a second group of the cells contacted with the formula (I)compound alone, or with the test compound alone. Effects that may beobserved include, but are not limited to, those described in theExamples section.

[M] In one aspect, pharmaceutical compositions are featured, whichinclude a compound of formula (I), or a pharmaceutically acceptable saltthereof (e.g., including any subgenera or specific compound thereof offormula (I), e.g., formula (I-A)) and a pharmaceutically acceptablecarrier. In some embodiments, the compositions can include one or moreadditional therapeutic agents (e.g., anticancer/chemotherapeutic agents)as defined anywhere herein.

In another aspect, methods of making the pharmaceutical compositionsdescribed herein are featured. In some embodiments, the methods includetaking any one or more of the compounds of formula (I) (e.g., includingany subgenera or specific compound thereof of formula (I), e.g., formula(I-A)) or a salt (e.g., a pharmaceutically acceptable salt) thereof asdefined anywhere herein, and mixing said compound(s) with one or morepharmaceutically acceptable carriers.

[N] In one aspect, methods of making the compounds described herein arefeatured. In some embodiments, the methods include taking any one of theintermediate compounds described herein and reacting it with one or morechemical reagents in one or more steps to produce a compound of formula(I) (and/or a compound of any of the other formulae described herein) ora salt (e.g., a pharmaceutically acceptable salt) thereof as definedanywhere herein.

[O] In one aspect, the compounds of formula (I) themselves (e.g.,including any subgenera or specific compound thereof of formula (I)) ora salt (e.g., a pharmaceutically acceptable salt) thereof as definedanywhere herein are featured. In another aspect, any of the formula (I)compounds specifically described herein are featured.

In some embodiments, the compounds of formula (I) are other than thosedescribed in the following printed publications:

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Publ.(2009), 57 pp. CODEN: USXXCO US 20090163545 A1 20090625 CAN 151: 115085AN 2009: 875997 CAPLUS Commercially available compounds.

In some embodiments, the foregoing exclusions can be combined with anyone or more of the exclusions described in section [E] above.

[P] Embodiments can include any one or more of the following features.

X can contain 2 ring atoms independently selected from N, NH, N(C₁-C₃alkyl), O, and S; e.g., N and S or N and NH.

One of the two ring atoms can be independently selected from N, NH, andN(C₁-C₃ alkyl) (e.g., N), and the other ring atom is independentlyselected from N, NH, N(C₁-C₃ alkyl), O, and S (e.g., S or NH, e.g., S).

X can have formula X-1:

in which:

X′ is NH, O, or S; and

one of X″ and X′″ is Y, and the other of X″ and X′″ is H or R^(a).

X′ can be S.

X′ can be NH.

X′″ can be Y.

X″ can be H or R^(a).

X″ can be H.

X″ can be R^(a). In embodiments, R^(a) can be C₁-C₈ alkyl (e.g., CH₃).In other embodiments, R^(a) can be phenyl that is optionally substitutedwith from 1-4 R^(e); or C₃-C₈ cycloalkyl which is optionally substitutedwith from 1-4 independently selected C₁-C₄ alkyl groups.

Y can be C₆-C₁₀ aryl (e.g., phenyl), which is optionally substitutedwith from 1-5 independently selected R^(b). In embodiments, Y can beunsubstituted phenyl.

R¹ can be C₆-C₁₀ aryl, which is optionally substituted with from 1-5independently selected R^(c).

R¹ can be phenyl, which is substituted with from 1-5 (e.g., 1-3, 1-2,or 1) independently selected R^(c).

Each occurrence of R^(c) can be, independently, selected from any onethe substituents delineated collectively in (aaa), (bbb), (ccc), and(ddd) below:

(aaa) C₁-C₆ alkoxy, C₁-C₆ alkyl, C₁-C₆ haloalkyl, —NH(C₁-C₆ alkyl),N(C₁-C₆ alkyl)₂, or —NHC(O)(C₁-C₆ alkyl), each of which is optionallysubstituted with —OH, —NH₂, or —SH;

(bbb) C(O)OH; —C(O)O(C₁-C₆ alkyl); —OC(O)(C₁-C₆ alkyl); —SO₂NH₂;—SO₂NH(C₁-C₆ alkyl); or —SO₂N(C₁-C₆ alkyl)₂; or—C(O)O—(CH₂)_(1-3(e.g.,1))—C(O)-(phenyl optionally substituted asdefined in (ddd) below (e.g., —C(O)O—CH₂—C(O)-(phenyl);

(ccc) C₃-C₆ cycloalkoxy or L-heterocyclyl containing from 5-7 ringatoms, wherein from 1-2 of the ring atoms of the heterocyclyl isindependently selected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl),NC(O)O(C₁-C₆ alkyl), O, and S; and each of which is optionallysubstituted with from 1-3 independently selected C₁-C₄ alkyl groups; andwherein L is a bond or C₁-C₆ alkylene; and

(ddd) phenyl or heteroaryl containing from 5-6 ring atoms, wherein from1-2 of the ring atoms of the heteroaryl is independently selected fromN, NH, N(C₁-C₃ alkyl), O, and S; wherein each of said phenyl andheteroaryl is optionally substituted with from 1-3 substituentsindependently selected from halo; hydroxyl; cyano; —C(O)(C₁-C₆ alkyl);C(O)OH; —C(O)O(C₁-C₆ alkyl); nitro; —NH₂; —NH(C₁-C₆ alkyl), N(C₁-C₆alkyl)₂, —NHC(O)(C₁-C₆ alkyl), C₁-C₆ alkoxy; C₁-C₆ haloalkoxy; C₁-C₆thioalkoxy; C₁-C₆ thiohaloalkoxy; C₁-C₆ alkyl, and C₁-C₆ haloalkyl,wherein said alkyl or alkyl portion is optionally substituted with —OH,—NH₂, or —SH.

In embodiments, each occurrence of R^(c) is, independently, selectedfrom:

-   -   C₁-C₆ alkoxy;    -   C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g., ethoxy); or C₁-C₆ alkoxy        (e.g., C₂-C₆ alkoxy, e.g., ethoxy) that is substituted with        —NH₂;    -   C₁-C₆ alkyl;    -   —NHC(O)(C₁-C₆ alkyl)    -   —C(O)OH;    -   L-heterocyclyl containing from 5-7 ring atoms, wherein from 1-2        of the ring atoms of the heterocyclyl is independently selected        from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), NC(O)O(C₁-C₆        alkyl), O, and S; and each of which is optionally substituted        with from 1-3 independently selected C₁-C₄ alkyl groups; and        wherein L is a bond or C₁-C₆ alkylene (e.g., a bond); e.g.,        R^(c) is optionally substituted morpholino or optionally        substituted piperazinyl; and    -   phenyl or heteroaryl containing from 5-6 ring atoms, wherein        from 1-2 of the ring atoms of the heteroaryl is independently        selected from N, NH, N(C₁-C₃ alkyl), O, and S; wherein each of        said phenyl and heteroaryl is optionally substituted with from        1-3 substituents independently selected from halo; hydroxyl;        cyano; —C(O)(C₁-C₆ alkyl); C(O)OH; —C(O)O(C₁-C₆ alkyl); nitro;        —NH₂; —NH(C₁-C₆ alkyl), N(C₁-C₆ alkyl)₂, —NHC(O)(C₁-C₆ alkyl),        C₁-C₆ alkoxy; C₁-C₆ haloalkoxy; C₁-C₆ thioalkoxy; C₁-C₆        thiohaloalkoxy; C₁-C₆ alkyl, and C₁-C₆ haloalkyl, wherein said        alkyl or alkyl portion is optionally substituted with —OH, —NH₂,        or —SH.

In embodiments, R^(c) can be C₁-C₆ alkoxy (e.g., ethoxy; e.g.,containing branched alkyl, such a iso-propoxy).

In embodiments, R^(c) can be C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g.,ethoxy; e.g., containing branched alkyl, such a iso-propoxy).

In embodiments, R^(c) can be C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g.,ethoxy; e.g., containing branched alkyl, such a iso-propoxy) that issubstituted with —NH₂.

R¹ can have formula A:

The following definitions apply to any formula described herein thatcontains formula (A).

One or two of R¹², R¹³, and R¹⁴ is(are) an independently selected R^(c),and the other(s) is(are) hydrogen.

R¹² can be R^(c) (as defined anywhere herein).

R¹³ can be H.

R¹⁴ can be H.

R¹⁴ can be R^(c).

R¹² can be R^(c) (as defined anywhere herein), and each of R¹³ and R¹⁴can be H.

R¹⁴ can be R^(c) (as defined anywhere herein), and each of R¹² and R¹³can be H.

In embodiments, R^(c) can be C₁-C₆ alkoxy (e.g., ethoxy; e.g.,containing branched alkyl, such a iso-propoxy).

In embodiments, R^(c) can be C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g.,ethoxy; e.g., containing branched alkyl, such a iso-propoxy).

In embodiments, R^(c) can be C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g.,ethoxy; e.g., containing branched alkyl, such a iso-propoxy) that issubstituted with —NH₂.

R^(c) can be —C(O)OH.

R^(c) can be L-heterocyclyl containing from 5-7 (e.g., 6) ring atoms,wherein from 1-2 of the ring atoms of the heterocyclyl is independentlyselected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), NC(O)O(C₁-C₆alkyl), O, and S; and each of which is optionally substituted with from1-3 independently selected C₁-C₄ alkyl groups; and wherein L is a bondor C₁-C₆ alkylene (e.g., a bond); e.g., R^(c) is morpholino orpiperazinyl. In embodiments, L can be a bond or CH₂ (e.g., L can be abond).

R¹³ can be H, and each of R¹² and R¹⁴ can be R^(c) (each independentlyas defined anywhere herein).

One of R¹² and R¹⁴ can be C₁-C₆ alkoxy (e.g., ethoxy), and the other ofR¹² and R¹⁴ can be independently selected from:

-   -   C₁-C₆ alkoxy;    -   C₁-C₆ alkyl;    -   —C(O)OH;    -   —NHC(O)(C₁-C₆ alkyl);    -   L-heterocyclyl containing from 5-7 ring atoms, wherein from 1-2        of the ring atoms of the heterocyclyl is independently selected        from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), NC(O)O(C₁-C₆        alkyl), O, and S; and each of which is optionally substituted        with from 1-3 independently selected C₁-C₄ alkyl groups; and        wherein L is a bond or C₁-C₆ alkylene (e.g., a bond); e.g.,        R^(c) is optionally substituted morpholino or optionally        substituted piperazinyl; and    -   phenyl or heteroaryl containing from 5-6 ring atoms, wherein        from 1-2 of the ring atoms of the heteroaryl is independently        selected from N, NH, N(C₁-C₃ alkyl), O, and S; wherein each of        said phenyl and heteroaryl is optionally substituted with from        1-3 substituents independently selected from halo; hydroxyl;        cyano; —C(O)(C₁-C₆ alkyl); C(O)OH; —C(O)O(C₁-C₆ alkyl); nitro;        —NH₂; —NH(C₁-C₆ alkyl), N(C₁-C₆ alkyl)₂, —NHC(O)(C₁-C₆ alkyl),        C₁-C₆ alkoxy; C₁-C₆ haloalkoxy; C₁-C₆ thioalkoxy; C₁-C₆        thiohaloalkoxy; C₁-C₆ alkyl, and C₁-C₆ haloalkyl, wherein said        alkyl or alkyl portion is optionally substituted with —OH, —NH₂,        or —SH.

R¹ can be heteroaryl, which contains from 5-10 ring atoms, wherein from1-4 of the ring atoms is independently selected from N, NH, N(C₁-C₃alkyl), O, and S; and wherein said heteroaryl ring is optionallysubstituted with from 1-3 independently selected R^(c).

R¹ can be heteroaryl, which contains from 5-6 ring atoms, wherein from1-4 (e.g., 1-2) of the ring atoms is independently selected from N, NH,N(C₁-C₃ alkyl), O, and S; and wherein said heteroaryl ring is optionallysubstituted with from 1-3 (e.g., 1-2 or 1) independently selected R^(c).For example, R¹ can be thiazolyl.

R¹ can be heteroaryl, which contains from 8-10 ring atoms, wherein from1-4 (e.g., 1-2) of the ring atoms is independently selected from N, NH,N(C₁-C₃ alkyl), O, and S; and wherein said heteroaryl ring is optionallysubstituted with from 1-3 (e.g., 1-2 or 1) independently selected R^(c).For example, R¹ can be indazolyl.

R² can be C₁-C₈ alkyl. For example, R² can be CH₃.

R² can be phenyl that is optionally substituted with from 1-4 R^(e). Inembodiments, R² can be unsubstituted phenyl.

R² can be C₃-C₈ cycloalkyl which is optionally substituted with from 1-4independently selected C₁-C₄ alkyl groups.

R² is heteroaryl containing from 5-6 (e.g., 5) ring atoms, wherein from1-4 of the ring atoms is independently selected from N, NH, N(C₁-C₃alkyl), NC(O)(C₁-C₆ alkyl), O, and S; and wherein said heteroaryl isoptionally substituted with from 1-3 R^(e). For example, R² can befuranyl, thienyl, or thiazolyl.

A can be N.

In some embodiments:

A is N;

X contains 2 ring atoms independently selected from N, NH, N(C₁-C₃alkyl), O, and S, and one of the ring atoms is independently selectedfrom N, NH, and N(C₁-C₃ alkyl), and the other ring atom is independentlyselected from N, NH, N(C₁-C₃ alkyl), O, and S; and

R¹ is C₆-C₁₀ aryl, which is optionally substituted with from 1-5independently selected R^(c) (e.g., R¹ can be phenyl, which issubstituted with from 1-5 (e.g., 1-3, 1-2, or 1) independently selectedR^(c)).

In certain embodiments, the compound can have formula I-A:

wherein:

X′ is NH, O, or S;

one of X″ and X′″ is Y, and the other of X″ and X′″ is H or R^(a);

one or two of R¹², R¹³, and R¹⁴ is(are) an independently selected R^(c),and the other(s) is(are) hydrogen; and

R² can be as defined anywhere herein.

X′ can be S or NH (e.g., S), X′″ is Y, and X″ is H or R^(a) (e.g., X″can be H or C₁-C₃ alkyl, e.g., CH₃).

Y can be C₆-C₁₀ aryl, which is optionally substituted with from 1-5independently selected R^(b) (e.g., Y can be unsubstituted phenyl).

R¹² can be R^(c), and each of R¹³ and R¹⁴ is H; or R¹⁴ is R^(c), andeach of R¹² and R¹³ is H.

Each occurrence of R^(c) is, independently, selected from:

-   -   C₁-C₆ alkoxy;    -   C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g., ethoxy); or C₁-C₆ alkoxy        (e.g., C₂-C₆ alkoxy, e.g., ethoxy) that is substituted with        —NH₂;    -   C₁-C₆ alkyl;    -   —C(O)OH;    -   —NHC(O)(C₁-C₆ alkyl);    -   L-heterocyclyl containing from 5-7 (e.g., 6) ring atoms, wherein        from 1-2 of the ring atoms of the heterocyclyl is independently        selected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl),        NC(O)O(C₁-C₆ alkyl), O, and S; and each of which is optionally        substituted with from 1-3 independently selected C₁-C₄ alkyl        groups; and wherein L is a bond or C₁-C₆ alkylene (e.g., a        bond); e.g., R^(c) is an optionally substituted morpholino or        optionally substituted piperazinyl ring; and    -   phenyl or heteroaryl containing from 5-6 ring atoms, wherein        from 1-2 of the ring atoms of the heteroaryl is independently        selected from N, NH, N(C₁-C₃ alkyl), O, and S; wherein each of        said phenyl and heteroaryl is optionally substituted with from        1-3 substituents independently selected from halo; hydroxyl;        cyano; —C(O)(C₁-C₆ alkyl); C(O)OH; —C(O)O(C₁-C₆ alkyl); nitro;        —NH₂; —NH(C₁-C₆ alkyl), N(C₁-C₆ alkyl)₂, —NHC(O)(C₁-C₆ alkyl),        C₁-C₆ alkoxy; C₁-C₆ haloalkoxy; C₁-C₆ thioalkoxy; C₁-C₆        thiohaloalkoxy; C₁-C₆ alkyl, and C₁-C₆ haloalkyl, wherein said        alkyl or alkyl portion is optionally substituted with —OH, —NH₂,        or —SH.

R^(c) can be C₁-C₆ alkoxy (e.g., ethoxy or iso-propoxy); or R^(c) can be—COOH; or R^(c) can be morpholino or piperazinyl.

R^(c) can be C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g., ethoxy oriso-propoxy) that is optionally substituted with —NH₂; or R^(c) can be—COOH; or R^(c) can be an optionally substituted morpholino oroptionally substituted piperazinyl ring.

R^(c) can be C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g., ethoxy oriso-propoxy) that is optionally substituted with —NH₂.

R² can be C₁-C₈ alkyl (e.g., CH₃).

R² can be phenyl or heteroaryl containing from 5-6 (e.g., 5) ring atoms,wherein from 1-4 of the ring atoms is independently selected from N, NH,N(C₁-C₃ alkyl), NC(O)(C₁-C₆ alkyl), O, and S; and wherein saidheteroaryl is optionally substituted with from 1-3 R^(e) (e.g., R² isheteroaryl as defined above, e.g., thienyl, furanyl, or thiazolyl).

X″ can be H or CH₃.

R² can be C₁-C₄ alkyl, such as CH₃. R² can be unsubstituted phenyl. R²can be heteroaryl containing from 5-6 ring atoms, wherein from 1-4 ofthe ring atoms is independently selected from N, NH, N(C₁-C₃ alkyl),NC(O)(C₁-C₆ alkyl), O, and S; and wherein said heteroaryl is optionallysubstituted with from 1-3 R^(e), such as thienyl, furanyl, or thiazolyl.

R¹² can be —C(O)OH. R¹² can be C₂-C₆ alkoxy that is optionallysubstituted with

-   -   NH₂, such as —OCH₂CH₃ or —OCH₂CH₂NH₂. R¹² can be heterocyclyl        containing from 5-7 ring atoms, wherein from 1-2 of the ring        atoms of the heterocyclyl is independently selected from N, NH,        N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), NC(O)O(C₁-C₆ alkyl), O, and        S; and each of which is optionally substituted with from 1-3        independently selected C₁-C₄ alkyl groups, such as an optionally        substituted piperazinyl ring.

X″ can be H or CH₃; and R² can be C₁-C₄ alkyl, such as CH₃; or R² can beunsubstituted phenyl; or R² can be heteroaryl containing from 5-6 ringatoms, wherein from 1-4 of the ring atoms is independently selected fromN, NH, N(C₁-C₃ alkyl), NC(O)(C₁-C₆ alkyl), O, and S; and wherein saidheteroaryl is optionally substituted with from 1-3 R^(e), such asthienyl, furanyl, or thiazolyl.

X″ can be H or CH₃; and

R² can be C₁-C₄ alkyl, such as CH₃; or R² can be unsubstituted phenyl;or R² can be heteroaryl containing from 5-6 ring atoms, wherein from 1-4of the ring atoms is independently selected from N, NH, N(C₁-C₃ alkyl),NC(O)(C₁-C₆ alkyl), O, and S; and wherein said heteroaryl is optionallysubstituted with from 1-3 R^(e), such as thienyl, furanyl, or thiazolyl;and

R¹² can be —C(O)OH; or R¹² can be C₂-C₆ alkoxy that is optionallysubstituted with —NH₂, such as —OCH₂CH₃ or —OCH₂CH₂NH₂; or R¹² can beheterocyclyl containing from 5-7 ring atoms, wherein from 1-2 of thering atoms of the heterocyclyl is independently selected from N, NH,N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), NC(O)O(C₁-C₆ alkyl), O, and S; andeach of which is optionally substituted with from 1-3 independentlyselected C₁-C₄ alkyl groups, such as an optionally substitutedpiperazinyl ring.

X″ can be H or CH₃; and

R² can be C₁-C₄ alkyl, such as CH₃; and

R¹² can be C₂-C₆ alkoxy that is optionally substituted with —NH₂, suchas —OCH₂CH₃ or —OCH₂CH₂NH₂;

In some embodiments:

A is N;

X contains 2 ring atoms independently selected from N, NH, N(C₁-C₃alkyl), O, and S, and one of the ring atoms is independently selectedfrom N, NH, and N(C₁-C₃ alkyl), and the other ring atom is independentlyselected from N, NH, N(C₁-C₃ alkyl), O, and S; and

R¹ is heteroaryl, which contains from 5-10 ring atoms, wherein from 1-4of the ring atoms is independently selected from N, NH, N(C₁-C₃ alkyl),O, and S; and wherein said heteroaryl ring is optionally substitutedwith from 1-3 independently selected W.

In certain embodiments, the compound can have formula I-B:

wherein:

X′ is NH, O, or S; and

one of X″ and X′″ is Y, and the other of X″ and X′″ is H or R^(a);

X′ can be S, X′″ can be Y, and X″ can be H or R^(a).

X″ can be H.

R¹ can be thiazolyl.

Y can be C₆-C₁₀ aryl, which is optionally substituted with from 1-5independently selected R^(b) (e.g., unsubstituted phenyl).

R² can be phenyl that is optionally substituted with from 1-4 R^(e); orheteroaryl containing from 5-6 (e.g., 5) ring atoms, wherein from 1-4 ofthe ring atoms is independently selected from N, NH, N(C₁-C₃ alkyl),NC(O)(C₁-C₆ alkyl), O, and S; and wherein said heteroaryl is optionallysubstituted with from 1-3 R^(e).

R² can be unsubstituted phenyl.

The compound can be selected from compounds delineated in FIGS. 11, 12,and 14.

The contacting can be in vitro.

The contacting is in vivo.

In some embodiments, any compound, composition, or method describedherein can also include or further include any one or more of the otherfeatures delineated in the detailed description and/or in the claims.

[Q] DEFINITIONS

The term “mammal” includes organisms, which include mice, rats, cows,sheep, pigs, rabbits, goats, horses, monkeys, dogs, cats, and humans.

In embodiments, an amount of a compound of formula (I) or salt thereofcan be an effective amount. “An effective amount” refers to an amount ofa compound that confers a therapeutic effect (e.g., treats, e.g.,controls, relieves, ameliorates, alleviates, or slows the progression ofor prevents, e.g., delays the onset of or reduces the risk ofdeveloping, a disease, disorder, or condition or symptoms thereof) onthe treated subject. The therapeutic effect may be objective (i.e.,measurable by some test or marker) or subjective (i.e., subject gives anindication of or feels an effect). An effective amount of the compounddescribed above may range from about 0.01 mg/kg to about 1000 mg/kg,(e.g., from about 0.1 mg/kg to about 100 mg/kg, from about 1 mg/kg toabout 100 mg/kg). Effective doses will also vary depending on route ofadministration, as well as the possibility of co-usage with otheragents.

The term “halo” or “halogen” refers to any radical of fluorine,chlorine, bromine or iodine.

In general, and unless otherwise indicated, substituent (radical) prefixnames are derived from the parent hydride by either (i) replacing the“ane” in the parent hydride with the suffixes “yl,” “diyl,” “triyl,”“tetrayl,” etc.; or (ii) replacing the “e” in the parent hydride withthe suffixes “yl,” “diyl,” “triyl,” “tetrayl,” etc. (Here the atom(s)with the free valence, when specified, is (are) given numbers as low asis consistent with any established numbering of the parent hydride).Accepted contracted names, e.g., adamantyl, naphthyl, anthryl,phenanthryl, furyl, pyridyl, isoquinolyl, quinolyl, and piperidyl, andtrivial names, e.g., vinyl, allyl, phenyl, and thienyl are also usedherein throughout. Conventional numbering/lettering systems are alsoadhered to for substituent numbering and the nomenclature of fused,bicyclic, tricyclic, and polycyclic rings.

The following definitions are used unless otherwise described. Specificand general values listed below for radicals, substituents, and rangesare for illustration only. They do not exclude other defined values orother values within defined ranges for the radicals and substituents.Unless otherwise indicated, alkyl, alkylene, alkoxy, alkenyl, and thelike denote both straight and branched groups.

The term “alkyl” refers to a saturated hydrocarbon chain that may be astraight chain or branched chain, containing the indicated number ofcarbon atoms. For example, C₁-C₆ alkyl indicates that the group may havefrom 1 to 6 (inclusive) carbon atoms in it. Any atom can be optionallysubstituted, e.g., by one or more substitutents. Examples of alkylgroups include, without limitation, methyl, ethyl, n-propyl, isopropyl,and tert-butyl.

As used herein, the term “C_(n-m) alkylene,” employed alone or incombination with other terms, refers to a non-branched divalent alkyllinking group having n to m carbon atoms.

The term “haloalkyl” refers to an alkyl group in which at least onehydrogen atom is replaced by halo. In some embodiments, more than onehydrogen atom (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) isreplaced by halo. In these embodiments, the hydrogen atoms can each bereplaced by the same halogen (e.g., fluoro) or the hydrogen atoms can bereplaced by a combination of different halogens (e.g., fluoro andchloro). “Haloalkyl” also includes alkyl moieties in which all hydrogenshave been replaced by halo (sometimes referred to herein asperhaloalkyl, e.g., perfluoroalkyl, such as trifluoromethyl). Any atomcan be optionally substituted, e.g., by one or more substituents.

As referred to herein, the term “alkoxy” refers to a group of formula—O(alkyl). Alkoxy can be, for example, methoxy (—OCH₃), ethoxy, propoxy,isopropoxy, butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy,2-pentoxy, 3-pentoxy, or hexyloxy. Likewise, the term “thioalkoxy”refers to a group of formula —S(alkyl). The terms “haloalkoxy” and“thio-haloalkoxy” refer to —O(haloalkyl) and —S(haloalkyl),respectively. Finally, the terms “cycloalkoxy” and “heterocyclyloxy”refer to a group of the formula —O(cycloalkyl) and —O(heterocyclyl),respectively.

The term “alkenyl” refers to a straight or branched hydrocarbon chaincontaining the indicated number of carbon atoms and having one or morecarbon-carbon double bonds. Any atom can be optionally substituted,e.g., by one or more substituents. Alkenyl groups can include, e.g.,vinyl, allyl, 1-butenyl, and 2-hexenyl. One of the double bond carbonscan optionally be the point of attachment of the alkenyl substituent.

The term “alkynyl” refers to a straight or branched hydrocarbon chaincontaining the indicated number of carbon atoms and having one or morecarbon-carbon triple bonds. Alkynyl groups can be optionallysubstituted, e.g., by one or more substituents. Alkynyl groups caninclude groups such as ethynyl, propargyl, and 3-hexynyl. One of thetriple bond carbons can optionally be the point of attachment of thealkynyl substituent.

The term “heterocyclyl” refers to a fully saturated monocyclic,bicyclic, tricyclic or other polycyclic ring system having one or moreconstituent heteroatom ring atoms independently selected from O, N (itis understood that one or two additional groups may be present tocomplete the nitrogen valence and/or form a salt), or S. The heteroatomor ring carbon can be the point of attachment of the heterocyclylsubstituent to another moiety. Any atom can be optionally substituted,e.g., by one or more substituents. Heterocyclyl groups can includegroups such as tetrahydrofuryl, tetrahydropyranyl, piperidyl(piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, andpyrrolidinyl. By way of example, a phrase such as “heterocyclic ringcontaining from 5-6 ring atoms”, wherein from 1-2 of the ring atoms isindependently selected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl),O, and S; and wherein said heterocyclic ring is optionally substitutedwith from 1-3 independently selected R^(a) would include (but not belimited to) tetrahydrofuryl, tetrahydropyranyl, piperidyl (piperidino),piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl.

The term “cycloalkyl” refers to a fully saturated monocyclic, bicyclic,tricyclic, or other polycyclic hydrocarbon group. Any atom can beoptionally substituted, e.g., by one or more substituents. A ring carbonserves as the point of attachment of a cycloalkyl group to anothermoiety. Cycloalkyl moieties can include groups such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, andnorbornyl (bicyclo[2.2.1]heptyl).

The term “cycloalkenyl” refers to partially unsaturated monocyclic,bicyclic, tricyclic, or other polycyclic hydrocarbon groups. A ringcarbon (e.g., saturated or unsaturated) is the point of attachment ofthe cycloalkenyl substituent. Any atom can be optionally substitutede.g., by one or more substituents. Cycloalkenyl moieties can include,e.g., cyclohexenyl, cyclohexadienyl, or norbornenyl.

The term “aryl” refers to an aromatic monocyclic, bicyclic (2 fusedrings), tricyclic (3 fused rings), or polycyclic (>3 fused rings)hydrocarbon ring system. One or more ring atoms can be optionallysubstituted by one or more substituents for example. Aryl moietiesinclude groups such as phenyl and naphthyl.

The term “heteroaryl” refers to an aromatic monocyclic, bicyclic (2fused rings), tricyclic (3 fused rings), or polycyclic (>3 fused rings)hydrocarbon groups having one or more heteroatom ring atomsindependently selected from O, N (it is understood that one or twoadditional groups may be present to complete the nitrogen valence and/orform a salt), or S. One or more ring atoms can be optionallysubstituted, e.g., by one or more substituents. Examples of heteroarylgroups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl,4H-quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl,β-carbolinyl, carbazolyl, coumarinyl, chromenyl, cinnolinyl,dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl,indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl,isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl, perimidinyl,phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl,phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl,purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl,pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, andxanthenyl.

As used herein, the descriptor “—CN” represents the cyano group (andvice versa), wherein the carbon and nitrogen atoms are bound together bya triple bond. As used herein, the descriptor “—OH” represents thehydroxy group (and vice versa). The descriptors “C═O” or “C(O)” refersto a carbon atom that is doubly bonded to an oxygen atom.

In general, when a definition for a particular variable includeshydrogen and non-hydrogen (halo, alkyl, aryl, etc.) possibilities, theterm “substituent(s) other than hydrogen” refers collectively to thenon-hydrogen possibilities for that particular variable.

The term “substituent” refers to a group “substituted” on groups such asan alkyl, haloalkyl, cycloakyl, heterocyclyl, aryl, or heteroaryl groupat any atom of that group. In one aspect, the substituent(s) on a groupare independently any one single or any combination of two or more ofthe permissible atoms or groups of atoms delineated for thatsubstituent. In another aspect, a substituent may itself be substitutedwith any one of the above substituents.

Further, as used herein, the phrase “optionally substituted” meansunsubstituted (e.g., substituted with hydrogen (H)) or substituted. Asused herein, the term “substituted” means that a hydrogen atom isremoved and replaced by a substituent. It is understood thatsubstitution at a given atom is limited by valency.

Descriptors such as “C₆-C₁₀ aryl that is optionally substituted withfrom 1-4 independently selected R^(b) (and the like) is intended toinclude both an unsubstituted C₆-C₁₀ aryl group and a C₆-C₁₀ aryl groupthat is substituted with from 1-4 independently selected R^(b). The useof a substituent (radical) prefix name such as alkyl without themodifier “optionally substituted” or “substituted” is understood to meanthat the particular substituent is unsubstituted. However, the use of“haloalkyl” without the modifier “optionally substituted” or“substituted” is still understood to mean an alkyl group, in which atleast one hydrogen atom is replaced by halo.

[R] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Methods and materialsare described herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a structural depiction of BAX, which shows that the BIM BH3trigger site on pro-apoptotic BAX (left hand side of structuraldepiction) localizes to the N-terminal face of the protein. In contrast,the canonical BH3 binding pocket of anti-apoptotic proteins (right handside of structural depiction) maps to the opposite side of BAX andremains occupied by the C-terminal helix 9 (yellow) when the protein isin the inactive, monomeric state.

FIG. 1B is a structural depiction of BAX, which shows that the BH3trigger site is comprised of a hydrophobic groove with a perimeter ofcharged and hydrophilic residues from α-helices 1 and 6. BAX is orientedto demonstrate its N-terminal face and the individual amino acids thatcomprise the trigger site, with the unstructured loop between α 1 and α2 depicted in the open position.

FIGS. 2A-2I are reaction schemes showing the syntheses used to prepare avariety of compounds of formula (I).

FIG. 3A is a flow diagram showing the results of a computationalscreening algorithm employing an in silico library of 750,000 smallmolecules docked on averaged minimized BAX structures. This screeningyielded a panel of 100 candidate BAX activator molecules (BAMs).

FIG. 3B is a compilation of the docked structures and demonstrates howcandidate BAMs occupy the topographic landscape of the BAX trigger site.

FIG. 4A is a graph showing the direct binding interaction betweenFITC-BIM SAHB and recombinant full-length BAX. This formed the basis fordeveloping a competitive fluorescence polarization binding assay toscreen for BAMs.

FIG. 4B is a graph showing that acetylated BIM SAHB (Ac-BIM SAHB), whicheffectively competed with FITC-BIM SAHB for BAX binding, served as apositive control for the assay.

FIG. 4C is a bar graph showing that eleven molecules achieved >55%displacement of FITC-BIM SAHB at the 100 μM screening dose. These elevencompounds were advanced to dose-responsive competitive binding analysis.*, no detectable FITC-BIM SAHB binding.

FIG. 4D is a graph that shows that a compound sometimes referred toherein as BAM7 emerged as the most effective of the tested smallmolecule competitors, displaying an IC₅₀ of 3.3 μM. Data are mean ands.d. for experiments performed in at least triplicate.

FIG. 4E shows the chemical structure of BAM7, which was confirmed by NMRand mass spectrometry, synthesized de novo, and found to have a similarIC₅₀ (4.4 μM) upon retesting by competitive FPA.

FIG. 4F shows the ¹H-NMR spectrum of BAM7.

FIG. 5A is a graph showing that in addition to engaging BAX, FITC-BIMSAHB binds to the broad range of anti-apoptotic targets.

FIGS. 5B-5D are graphs summarizing the following data. The specificityof BAM7 for the BH3 binding site on BAX was examined by competitive FPAemploying FITC-BIM SAHB and anti-apoptotic BCL-XL, MCL-1, and BFL-1/A1.Whereas Ac-BIM SAHB effectively competed with FITC-BIM SAHB for bindingto a diversity of anti-apoptotic targets, BAM7 demonstrated little to nocapacity to compete with FITC-BIM SAHB for interaction at the BH3binding sites of anti-apoptotic proteins. Data are mean and s.d. forexperiments performed in at least triplicate.

FIG. 6A shows (i) a graph in which the measured chemical shift changesof ¹⁵N-BAX upon BAM7 titration up to a ratio of 1:1 BAX:BAM7 are plottedas a function of BAX residue number and (ii) a ribbon diagram. Residueswith significant backbone amide chemical shift change are concentratedin the region of the trigger site (α1, α6; magenta). The Cα atoms ofaffected residues are represented as spheres in the ribbon diagram andlighter shaded bars in the graph (calculated significancethreshold >0.009 p.p.m.).

FIG. 6B is a structural depiction of the docked structure of BAM7 at thetrigger site and predicts that the pyrazolone core of BAM7 sits at thebase of the 6A7 activation epitope (amino acids 12-24), with themolecule's carbonyl group engaged in hydrogen bonding interactions withK21. The ethoxyphenyl group is positioned at the confluence of residuescomprising the α1-α2 loop's C-terminus and the N-termini□ of α1 and α2,a presumed hinge region for loop opening upon initiation of direct BAXactivation. The methyl and phenylthiazol R groups are predicted to makehydrophobic contact with aliphatic residues of α1 and α6, which alsoform a portion of the BIM BH3-binding groove.

FIG. 7 includes a ribbon diagram and a graph. BAM7 induces allostericchanges in key functional domains implicated in functional BAXactivation. Upon increasing the ratio of BAM7:¹⁵N-BAX from 1:1 to 2:1, aseries of chemical shift changes become more prominent in the α1-α2loop, α2 (BH3), and α9, three regions previously implicated in BIMBH3-triggered N-terminal loop opening, BAX BH3 exposure, and C-terminalhelix mobilization, respectively¹¹. These BAM7-induced allostericchanges reflect a major conformational change that has been linked tofunctional BAX activation. Cα atoms of affected residues are representedas spheres in the ribbon diagram and lighter shaded bars in the plot(calculated significance threshold >0.011 p.p.m.). The α1-α2 loop, α2(BH3), and α9 are highlighted in pink, cyan, and yellow, respectively.

FIG. 8A is a graph showing that Co-incubation of BAM7 (10, 20, and 30μM) and monomeric BAX (5 μM) induces dose- and time-responsive BAXoligomerization, as monitored by size exclusion chromatography.

FIG. 8B is a graph showing that in the presence of ANTS/DPX-loadedliposomes,

BAM7 treatment triggers dose-responsive BAX-mediated liposomal release.The exposure of liposomes to BAM7 or BAX alone had no such effect.

FIG. 8C is a bar graph that shows BAM7 selectively impaired theviability of Bak^(−/−) MEFs, but had no effect on MEFs that lack BAX(Bax^(−/−)) or both BAX and BAK (Bax^(−/−)Bak^(−/−)).

FIG. 8D shows that Bax^(−/−)Bak^(−/−) MEFs reconstituted with EGFP-BAX(˜60% EGFP-positive cells) display dose-responsive BAX translocationupon exposure to BAM7, as evidenced by the conversion of EGFP-BAXlocalization from a diffuse pattern to a mitochondrion-localizeddistribution. EGFP-BAX, green; Mitotracker, red; Colocalization, yellow;BAM7, 30 μM; Vehicle, 0.3% DMSO. Data are mean and s.d. for experimentsperformed in quadruplicate.

FIG. 8E shows Bak^(−/−) MEFs that contain endogenous BAX exhibiting themorphologic features of apoptosis in response to BAM7 treatment (15 μM).The time lapse images reveal progressive cellular shrinkage, membraneblebbing, and the formation of apoptotic bodies. 1, 20 min; 2, 6 h; 3,12 h; 4, 12.5 h; 5, 13.5 h, 6, 14.5 h; 7, 16.5 h; 8, 17.5 h.

FIG. 9A-9D are bar graphs that shows formula (I) compounds selectivelyimpair the viability of Bak^(−/−) MEFs, but had no effect on MEFs thatlack BAX (Bax^(−/−)) or both BAX and BAK (Bax^(−/−)Bak^(−/−)). For eachtriplet of bars centered on the indicated x-axis value, the BAK knockoutresult is the left-most bar; the BAX knockout result is the middle bar;and the double knockout result is the right-most bar.

FIG. 10A shows that BAM7 dose-responsively impairs the viability of DHL5diffuse large B-cell lymphoma (DLBCL) cells and the addition ofBCL-2/BCL-XL inhibitor ABT-737 further sensitizes the cells to BAM7anti-cancer activity.

FIGS. 10B and 10C are bar graphs showing that BAM7 can sensitize DHL5DLBCL cells to ABT-737, which is otherwise less effective in DHL5 cellsdue to the expression of anti-apoptotic proteins that lie outside itsbinding spectrum.

In FIGS. 10A-10C, for each triplet of bars centered on the indicatedx-axis value, the BAM7 (10 μM) is the left-most bar; the ABT-737 is themiddle bar; and BAM7 (10 μM) plus ABT-737 is the right-most bar.

FIG. 11 includes the chemical structures and binding data for formula(I) compounds.

FIG. 12 includes the chemical structures of some formula (I) compounds.

FIG. 13 includes data that supplements the screening data provided inFIG. 4C.

FIG. 14 includes the chemical structures of some formula (I) compoundsthat are encompassed by formula (I-A) or (I-B).

FIGS. 15A-15H are graphs that demonstrate the anti-leukemic activity ofBAM7.

FIG. 16 is a graph that compares the anti-leukemic activity of BAM7 tocompounds 165-93, 165-60, and 172-90 (see FIG. 14).

FIG. 17 is a graph that demonstrates the broad anti-leukemic activity ofcompound 172-90.

FIGS. 18A and 18B are graphs that demonstrate that compound 172-90overcomes the apoptotic resistance conferred by BCL-2 familyanti-apoptotic members BCL-XL and MCL-1; whereas the BCL-2/BCL-XLselective inhibitor ABT-737 induces cell death of the BCL-XL-dependentleukemia cell line, significant resistance to ABT-737 is manifest in theisogenic MCL-1 dependent leukemia cell line. In contrast, FIGS. 18C and18D are graphs that demonstrate that compound 172-90 inducesdose-responsive caspase 3/7 activity and cell death in both cell lines,overcoming formidable apoptotic resistance.

FIGS. 19A-19F are reaction schemes showing the syntheses used to preparea variety of compounds of formula (I), such as those delineated in FIG.14.

DETAILED DESCRIPTION

This application features pyrazol-3-one compounds that activate apro-apoptotic function of BAX, making them therapeutically useful fortreating (e.g., controlling, relieving, ameliorating, alleviating, orslowing the progression of) or preventing (e.g., delaying the onset ofor reducing the risk of developing) diseases, disorders, and conditionsassociated with deregulated apoptosis of cells (e.g., diseased ordamaged cells; e.g., insufficient apoptosis of diseased or damagedcells; or lack of or reduced apoptosis of diseased or damaged cells).Examples of such diseases, disorders, and conditions include (but arenot limited to) those associated with blockade(s) of cell death pathways(e.g., over-expression of anti-apoptotic BCL-2 proteins), e.g.,hyperproliferative diseases, such as cancer. While not wishing to bebound by theory, it is believed that the compounds described hereininduce and increase apoptosis in target cells (e.g., pathogenic cellsincluding, but not limited to, cancer cells), thereby suppressing tumorgrowth and/or proliferation. It is further believed that increasingapoptosis in said target cells reestablishes the normal apoptoticcontrol that, during homeostasis, is associated with a regulated balancebetween pro- and anti-apoptotic protein functions.

Compounds

This application features pyrazol-3-one compounds having formula (I)below as well as compositions and methods that include the formula (I)compounds.

Here and throughout this specification, the attendant variables R¹, R²,A, X, and Y (and any sub-variables) can be as defined anywhere herein.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, can also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention which are, for brevity, described in thecontext of a single embodiment, can also be provided separately or inany suitable sub-combination.

Thus, for ease of exposition, it is also understood that where in thisspecification, a variable (e.g., R¹) is defined by “as defined anywhereherein” (or the like), the definitions for that particular variableinclude the first occurring and broadest generic definition as well asany sub-generic and specific definitions delineated anywhere in thisspecification.

Variable X

In some embodiments, X is heteroaryl, which contains 5 ring atoms,wherein from 1-2 of the ring atoms is/are independently selected from N,NH, N(C₁-C₃ alkyl), O, and S; wherein:

-   -   X is connected to the pyrazolone nitrogen via a ring carbon atom        in X; and    -   X is optionally further substituted with 1 R^(a):

or

X is phenyl optionally substituted with from 1-5 R^(a).

In some embodiments, X is heteroaryl, which contains 5 ring atoms,wherein from 1-2 of the ring atoms is/are independently selected from N,NH, N(C₁-C₃ alkyl), O, and S; wherein:

-   -   X is connected to the pyrazolone nitrogen via a ring carbon atom        in X; and    -   X is optionally further substituted with 1 R^(a):

In some embodiments, X contains 2 ring atoms independently selected fromN,

NH, N(C₁-C₃ alkyl), O, and S (e.g., selected from N and S; e.g.,selected from N and NH).

In certain embodiments, one of the two ring atoms is independentlyselected from N, NH, and N(C₁-C₃ alkyl) (e.g., N), and the other ringatom is independently selected from N, NH, N(C₁-C₃ alkyl), O, and S(e.g., the other ring atom is O or S, e.g., S; or e.g., NH).

As an example, X can have formula X-1:

in which:

X′ is NH, O, or S; and

one of X″ and X′″ is Y, and the other of X″ and X′″ is H or R^(a).

Embodiments in which X has formula X-1 can include one or more of thefollowing features.

X′ is S. X′ is NH.

X′″ is Y, which can be as defined anywhere herein, and X″ is H or R^(a).

X′ is S; and X′″ is Y, which can be as defined anywhere herein, and X″is H or R^(a) (e.g., X″ is H).

X′ is NH; and X′″ is Y, which can be as defined anywhere herein, and X″is H or R^(a) (e.g., X″ is H).

X″ is H.

X″ is R^(a). In certain embodiments, R^(a) is C₁-C₈ (e.g., C₁-C₆,C₁-C₃)alkyl (e.g., CH₃). In other embodiments, R^(a) is phenyl that isoptionally substituted with from 1-4 R^(e). In still other embodiments,R^(a) is or C₃-C₈ cycloalkyl which is optionally substituted with from1-4 independently selected C₁-C₄ alkyl groups.

X″ is Y, which can be as defined anywhere herein; and X′″ is H or R^(a).In embodiments, X′ is S or NH (e.g., S).

Variable Y

In some embodiments, Y is:

(i) C₆-C₁₀ aryl, which is optionally substituted with from 1-5independently selected R^(b); or

(ii) heteroaryl, which contains from 5-10 ring atoms, wherein from 1-4of the ring atoms is independently selected from N, NH, N(C₁-C₃ alkyl),O, and S; and wherein said heteroaryl ring is optionally substitutedwith from 1-3 independently selected R^(b); or

(iii) C₁-C₆ alkyl or C₁-C₆ haloalkyl, each of which is optionallysubstituted with —OH, —NH₂, or —SH.

In some embodiments, Y is C₆-C₁₀ aryl, which is optionally substitutedwith from 1-5 independently selected R^(b). In certain embodiments, Y isphenyl, which is optionally substituted with from 1-5 independentlyselected R^(b). In certain embodiments, Y is unsubstituted phenyl.

In some embodiments, Y is heteroaryl, which contains from 5-10 ringatoms, wherein from 1-4 of the ring atoms is independently selected fromN, NH, N(C₁-C₃ alkyl), O, and S; and wherein said heteroaryl ring isoptionally substituted with from 1-3 independently selected R^(b).

In some embodiments, Y is heteroaryl, which contains from 5-6 ringatoms, wherein from 1-4 (e.g., 1-2) of the ring atoms is independentlyselected from N, NH, N(C₁-C₃ alkyl), O, and S; and wherein saidheteroaryl ring is optionally substituted with from 1-3 independentlyselected R^(b).

In some embodiments, Y is C₁-C₆ alkyl or C₁-C₆ haloalkyl, each of whichis optionally substituted with —OH, —NH₂, or —SH (e.g., C₁-C₆ alkyl,which is optionally substituted with —OH, —NH₂, or —SH; e.g., C₁-C₆alkyl, such as CH₃).

Variable R¹

R¹ is:

(i) C₆-C₁₀ aryl, which is optionally substituted with from 1-5independently selected R^(c); or

(ii) heteroaryl, which contains from 5-10 ring atoms, wherein from 1-4of the ring atoms is independently selected from N, NH, N(C₁-C₃ alkyl),O, and S; and wherein said heteroaryl ring is optionally substitutedwith from 1-3 independently selected R^(c); or

(iii) —C(O)—(C₆-C₁₀ aryl or heteroaryl, which contains from 5-10 ringatoms as defined in R¹ definition (i) and (ii), respectively); or

(iv) hydrogen.

In some embodiments, R¹ is any one, two, or three of the above (e.g.,(i) and/or (ii) and one of (iii) or (iv); e.g., (i) and/or (ii); e.g.,(iii) and/or (iv)).

In some embodiments, R¹ is C₆-C₁₀ aryl, which is optionally substitutedwith from 1-5 independently selected R^(c).

In certain embodiments, R¹ is phenyl, which is substituted with from 1-5(e.g., 1-3, 1-2, or 1) independently selected R^(c).

In certain embodiments, each occurrence of R^(c) is, independently,selected from any one the substituents delineated collectively in (aaa),(bbb), (ccc), and (ddd) below:

(aaa) C₁-C₆ alkoxy, C₁-C₆ alkyl, C₁-C₆ haloalkyl, —NH(C₁-C₆ alkyl),N(C₁-C₆ alkyl)₂, or —NHC(O)(C₁-C₆ alkyl), each of which is optionallysubstituted with —OH, —NH₂, or —SH;

(bbb) C(O)OH; —C(O)O(C₁-C₆ alkyl); —OC(O)(C₁-C₆ alkyl); —SO₂NH₂;—SO₂NH(C₁-C₆ alkyl); or —SO₂N(C₁-C₆ alkyl)₂; or—C(O)O—(CH₂)_(1-3(e.g.,1))—C(O)-(phenyl optionally substituted asdefined in (ddd) below (e.g., —C(O)O—CH₂—C(O)-(phenyl);

(ccc) C₃-C₆ cycloalkoxy or L-heterocyclyl containing from 5-7 ringatoms, wherein from 1-2 of the ring atoms of the heterocyclyl isindependently selected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl),NC(O)O(C₁-C₆ alkyl), O, and S; and each of which is optionallysubstituted with from 1-3 independently selected C₁-C₄ alkyl groups; andwherein L is a bond or C₁-C₆ alkylene (e.g., a bond); e.g., R^(c) ismorpholino or piperazinyl; and

(ddd) phenyl or heteroaryl containing from 5-6 ring atoms, wherein from1-2 of the ring atoms of the heteroaryl is independently selected fromN, NH, N(C₁-C₃ alkyl), O, and S; wherein each of said phenyl andheteroaryl is optionally substituted with from 1-3 substituentsindependently selected from halo; hydroxyl; cyano; —C(O)(C₁-C₆ alkyl);C(O)OH; —C(O)O(C₁-C₆ alkyl); nitro; —NH₂; —NH(C₁-C₆ alkyl), N(C₁-C₆alkyl)₂, —NHC(O)(C₁-C₆ alkyl), C₁-C₆ alkoxy; C₁-C₆ haloalkoxy; C₁-C₆thioalkoxy; C₁-C₆ thiohaloalkoxy; C₁-C₆ alkyl, and C₁-C₆ haloalkyl,wherein said alkyl or alkyl portion is optionally substituted with —OH,—NH₂, or —SH.

In certain embodiments, each occurrence of R^(c) is, independently,selected from:

-   -   C₁-C₆ alkoxy;    -   C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g., ethoxy); or C₁-C₆ alkoxy        (e.g., C₂-C₆ alkoxy, e.g., ethoxy) that is substituted with        —NH₂;    -   C₁-C₆ alkyl;    -   —NHC(O)(C₁-C₆ alkyl)    -   —C(O)OH;    -   L-heterocyclyl containing from 5-7 (e.g., 5-6, or 6) ring atoms,        wherein from 1-2 of the ring atoms of the heterocyclyl is        independently selected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆        alkyl), NC(O)O(C₁-C₆ alkyl), O, and S; and each of which is        optionally substituted with from 1-3 independently selected        C₁-C₄ alkyl groups; and wherein L is a bond or C₁-C₆ alkylene        (e.g., a bond); e.g., R^(c) is morpholino or piperazinyl; and    -   phenyl or heteroaryl containing from 5-6 ring atoms, wherein        from 1-2 of the ring atoms of the heteroaryl is independently        selected from N, NH, N(C₁-C₃ alkyl), O, and S; wherein each of        said phenyl and heteroaryl is optionally substituted with from        1-3 substituents independently selected from halo; hydroxyl;        cyano; —C(O)(C₁-C₆ alkyl); C(O)OH; —C(O)O(C₁-C₆ alkyl); nitro;        —NH₂; —NH(C₁-C₆ alkyl), N(C₁-C₆ alkyl)₂, —NHC(O)(C₁-C₆ alkyl),        C₁-C₆ alkoxy; C₁-C₆ haloalkoxy; C₁-C₆ thioalkoxy; C₁-C₆        thiohaloalkoxy; C₁-C₆ alkyl, and C₁-C₆ haloalkyl, wherein said        alkyl or alkyl portion is optionally substituted with —OH, —NH₂,        or —SH.

In certain embodiments, R^(c) is C₁-C₆ alkoxy (e.g., ethoxy e.g.,containing branched alkyl, such as iso-propoxy).

In embodiments, R^(c) can be C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g.,ethoxy; e.g., containing branched alkyl, such a iso-propoxy).

In embodiments, R^(c) can be C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g.,ethoxy; e.g., containing branched alkyl, such a iso-propoxy) that issubstituted with —NH₂.

In certain embodiments, R¹ has formula A:

wherein one or two of R¹², R¹³, and R¹⁴ is(are) an independentlyselected R^(c), and the other(s) is(are) hydrogen.

Embodiments in which R¹ has formula A can include one or more of thefollowing features.

R¹² is R^(c).

R¹³ is H.

R14 is H.

R¹⁴ is R^(c).

R¹² is R^(c), and each of R¹³ and R¹⁴ is H.

R¹⁴ is R^(c), and each of R¹² and R¹³ is H.

In some of the above described embodiments, R^(c) is C₁-C₆ alkoxy (e.g.,ethoxy or iso-propoxy).

In embodiments, R^(c) can be C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g.,ethoxy; e.g., containing branched alkyl, such a iso-propoxy).

In embodiments, R^(c) can be C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g.,ethoxy; e.g., containing branched alkyl, such a iso-propoxy) that issubstituted with —NH₂. In some of the above described embodiments, R^(c)is —C(O)OH (or a salt thereof, e.g., sodium salt thereof).

In some of the above described embodiments, R^(c) is L-heterocyclylcontaining from 5-7 (e.g., 6) ring atoms, wherein from 1-2 of the ringatoms of the heterocyclyl is independently selected from N, NH, N(C₁-C₆alkyl), NC(O)(C₁-C₆ alkyl), NC(O)O(C₁-C₆ alkyl), O, and S; and each ofwhich is optionally substituted with from 1-3 independently selectedC₁-C₄ alkyl groups; and wherein L is a bond or C₁-C₆ alkylene (e.g., Lis a bond or CH₂; e.g., a bond); e.g., R^(c) is morpholino orpiperazinyl.

R¹³ is H, and each of R¹² and R¹⁴ is R^(c). In certain embodiments, oneof R¹² and R¹⁴ is C₁-C₆ alkoxy (e.g., ethoxy), and the other of R¹² andR¹⁴ is independently selected from:

-   -   C₁-C₆ alkoxy;    -   C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g., ethoxy); or C₁-C₆ alkoxy        (e.g., C₂-C₆ alkoxy, e.g., ethoxy) that is substituted with        —NH₂;    -   C₁-C₆ alkyl;    -   —C(O)OH;    -   —NHC(O)(C₁-C₆ alkyl);    -   L-heterocyclyl containing from 5-7 ring atoms, wherein from 1-2        of the ring atoms of the heterocyclyl is independently selected        from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), NC(O)O(C₁-C₆        alkyl), O, and S; and each of which is optionally substituted        with from 1-3 independently selected C₁-C₄ alkyl groups; and        wherein L is a bond or C₁-C₆ alkylene (e.g., a bond); e.g.,        R^(c) is morpholino or piperazinyl; and    -   phenyl or heteroaryl containing from 5-6 ring atoms, wherein        from 1-2 of the ring atoms of the heteroaryl is independently        selected from N, NH, N(C₁-C₃ alkyl), O, and S; wherein each of        said phenyl and heteroaryl is optionally substituted with from        1-3 substituents independently selected from halo; hydroxyl;        cyano; —C(O)(C₁-C₆ alkyl); C(O)OH; —C(O)O(C₁-C₆ alkyl); nitro;        —NH₂; —NH(C₁-C₆ alkyl), N(C₁-C₆ alkyl)₂, —NHC(O)(C₁-C₆ alkyl),        C₁-C₆ alkoxy; C₁-C₆ haloalkoxy; C₁-C₆ thioalkoxy; C₁-C₆        thiohaloalkoxy; C₁-C₆ alkyl, and C₁-C₆ haloalkyl, wherein said        alkyl or alkyl portion is optionally substituted with —OH, —NH₂,        or —SH.

In some embodiments, R¹ is heteroaryl, which contains from 5-10 ringatoms, wherein from 1-4 of the ring atoms is independently selected fromN, NH, N(C₁-C₃ alkyl), O, and S; and wherein said heteroaryl ring isoptionally substituted with from 1-3 independently selected R^(c).

In certain embodiments, R¹ is heteroaryl, which contains from 5-6 ringatoms, wherein from 1-4 (e.g., 1-2) of the ring atoms is independentlyselected from N, NH, N(C₁-C₃ alkyl), O, and S; and wherein saidheteroaryl ring is optionally substituted with from 1-3 (e.g., 1-2 or 1)independently selected R^(c). For example, R¹ can be thiazolyl.

In other embodiments, R¹ is heteroaryl, which contains from 8-10 ringatoms, wherein from 1-4 (e.g., 1-2) of the ring atoms is independentlyselected from N, NH, N(C₁-C₃ alkyl), O, and S; and wherein saidheteroaryl ring is optionally substituted with from 1-3 (e.g., 1-2 or 1)independently selected R^(c). For example, R¹ can be indazolyl.

In some embodiments, R¹ is hydrogen.

In some embodiments, R¹ is —C(O)—(C₆-C₁₀ aryl or heteroaryl, whichcontains from 5-10 ring atoms as defined in R¹ definition (i) and (ii),respectively); e.g., —C(O)-(heteroaryl, which contains from 5-10 (e.g.,5-6, e.g., 5) ring atoms as defined in R¹ definition (i) and (ii),respectively), such as —C(O)-(thiazolyl).

Variable R²

In some embodiments, R² is C₁-C₈ alkyl (e.g., C₁-C₆ alkyl, e.g., C₁-C₃alkyl, e.g., CH₃).

In some embodiments, R² is phenyl that is optionally substituted withfrom 1-4 R^(e). In certain embodiments, R² is unsubstituted phenyl.

In some embodiments, R² is heteroaryl containing from 5-6 (e.g., 5) ringatoms, wherein from 1-4 of the ring atoms is independently selected fromN, NH, N(C₁-C₃ alkyl), NC(O)(C₁-C₆ alkyl), O, and S; and wherein saidheteroaryl is optionally substituted with from 1-3 R^(e). In certainembodiments, R² is heteroaryl containing from 5 ring atoms, wherein from1-4 of the ring atoms is independently selected from N, NH, N(C₁-C₃alkyl), NC(O)(C₁-C₆ alkyl), O, and S; and wherein said heteroaryl isoptionally substituted with from 1-3 R^(e); e.g., theinyl or furyl.

In some embodiments, R² is C₃-C⁸ cycloalkyl which is optionallysubstituted with from 1-4 independently selected C₁-C₄ alkyl groups.

Variable A

In some embodiments, A is N.

Non-Limiting Combinations of Attendant Variables

In some embodiments:

A is N;

X contains 2 ring atoms independently selected from N, NH, N(C₁-C₃alkyl), O, and S, and one of the ring atoms is independently selectedfrom N, NH, and N(C₁-C₃ alkyl) (e.g., N), and the other ring atom isindependently selected from N, NH, N(C₁-C₃ alkyl), O, and S (e.g., theother ring atom is S or NH, e.g., S); and

R¹ is C₆-C₁₀ aryl, which is optionally substituted with from 1-5independently selected R^(c) (e.g., R¹ is phenyl, which is substitutedwith from 1-5 (e.g., 1-3, 1-2, or 1) independently selected R^(c)),which R^(c) can be as defined anywhere herein.

In some embodiments the compound has formula I-A:

wherein:

X′ is NH, O, or S (e.g., S or NH, e.g., S);

one of X″ and X′″ is Y, and the other of X″ and X′″ is H or R^(a); and

one or two of R¹², R¹³, and R¹⁴ is(are) an independently selected R^(c),and the other(s) is(are) hydrogen; R² can be as defined anywhere herein.

Embodiments in which the compound has formula I-A can include one ormore of the following features.

X′ is S, X′″ is Y, and X″ is H or R^(a) (e.g., X″ is H).

X′ is NH, X′″ is Y, and X″ is H or R^(a) (e.g., X″ is H).

R^(a) is C₁-C₈ alkyl (e.g., C₁-C₆ alkyl, e.g., C₁-C₃ alkyl, e.g., CH₃).

Y is C₆-C₁₀ aryl, which is optionally substituted with from 1-5independently selected R^(b) (e.g., Y is unsubstituted phenyl).

R¹² is R^(c), and each of R¹³ and R¹⁴ is H; or R¹⁴ is R^(c), and each ofR¹² and R¹⁴ is H.

Each occurrence of R^(c) is, independently, selected from:

-   -   C₁-C₆ alkoxy;    -   C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g., ethoxy); or C₁-C₆ alkoxy        (e.g., C₂-C₆ alkoxy, e.g., ethoxy) that is substituted with        —NH₂;    -   C₁-C₆ alkyl;    -   —C(O)OH;    -   —NHC(O)(C₁-C₆ alkyl);    -   L-heterocyclyl containing from 5-7 ring atoms, wherein from 1-2        of the ring atoms of the heterocyclyl is independently selected        from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), NC(O)O(C₁-C₆        alkyl), O, and S; and each of which is optionally substituted        with from 1-3 independently selected C₁-C₄ alkyl groups; and        wherein L is a bond or C₁-C₆ alkylene (e.g., a bond); e.g.,        R^(c) is morpholino or piperidinyl; and    -   phenyl or heteroaryl containing from 5-6 ring atoms, wherein        from 1-2 of the ring atoms of the heteroaryl is independently        selected from N, NH, N(C₁-C₃ alkyl), O, and S; wherein each of        said phenyl and heteroaryl is optionally substituted with from        1-3 substituents independently selected from halo; hydroxyl;        cyano; —C(O)(C₁-C₆ alkyl); C(O)OH; —C(O)O(C₁-C₆ alkyl); nitro;        —NH₂; —NH(C₁-C₆ alkyl), N(C₁-C₆ alkyl)₂, —NHC(O)(C₁-C₆ alkyl),        C₁-C₆ alkoxy; C₁-C₆ haloalkoxy; C₁-C₆ thioalkoxy; C₁-C₆        thiohaloalkoxy; C₁-C₆ alkyl, and C₁-C₆ haloalkyl, wherein said        alkyl or alkyl portion is optionally substituted with —OH, —NH₂,        or —SH.

R^(c) is C₁-C₆ alkoxy (e.g., ethoxy; e.g., containing branched alkyl,such as iso-propoxy).

R^(c) is C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g., ethoxy; e.g.,containing branched alkyl, such a iso-propoxy).

R^(c) is C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g., ethoxy; e.g.,containing branched alkyl, such a iso-propoxy) that is substituted with—NH₂.

R² is C₁-C₈ alkyl (e.g., C₁-C₆ alkyl, e.g., C₁-C₃ alkyl, e.g., CH₃). R²is phenyl that is optionally substituted with from 1-4 R^(e). R² isheteroaryl containing from 5-6 (e.g., 5) ring atoms, wherein from 1-4 ofthe ring atoms is independently selected from N, NH, N(C₁-C₃ alkyl),NC(O)(C₁-C₆ alkyl), O, and S; and wherein said heteroaryl is optionallysubstituted with from 1-3 W.

In some embodiments of formula I-A:

-   -   X′ is S or NH;    -   X′″ is Y;    -   X″ is H or R^(a); R^(a) is C₁-C₈ alkyl (e.g., C₁-C₆ alkyl, e.g.,        C₁-C₃ alkyl, e.g., CH₃); (e.g., X″ is H or CH₃; e.g., X″ is H);    -   Y is C₆-C₁₀ aryl, which is optionally substituted with from 1-5        independently selected R^(b) (e.g., Y is unsubstituted phenyl);        and    -   R² is C₁-C₈ alkyl (e.g., C₁-C₆ alkyl, e.g., C₁-C₃ alkyl, e.g.,        CH₃).

In certain of these formula I-A embodiments:

-   -   X′ is S or NH;    -   X′″ is Y;    -   X″ is H or R^(a); R^(a) is C₁-C₈ alkyl (e.g., C₁-C₆ alkyl, e.g.,        C₁-C₃ alkyl, e.g., CH₃); (e.g., X″ is H or CH₃; e.g., X″ is H);    -   Y is C₆-C₁₀ aryl, which is optionally substituted with from 1-5        independently selected R^(b) (e.g., Y is unsubstituted phenyl);    -   R² is C₁-C₈ alkyl (e.g., C₁-C₆ alkyl, e.g., C₁-C₃ alkyl, e.g.,        CH₃, and    -   R¹² is:    -   —C(O)OH; or    -   C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g., ethoxy; e.g., containing        branched alkyl, such a iso-propoxy) that is optionally        substituted with —NH₂, such as —OCH₂CH₃ or —OCH₂CH₂NH₂; or    -   heterocyclyl containing from 5-7 (e.g., 6) ring atoms, wherein        from 1-2 of the ring atoms of the heterocyclyl is independently        selected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl),        NC(O)O(C₁-C₆ alkyl), O, and S; and each of which is optionally        substituted with from 1-3 independently selected C₁-C₄ alkyl        groups, such as an optionally substituted piperazinyl ring.

In certain of these formula I-A embodiments:

-   -   X′ is S or NH;    -   X′″ is Y;    -   X″ is H or R^(a); R^(a) is C₁-C₈ alkyl (e.g., C₁-C₆ alkyl, e.g.,        C₁-C₃ alkyl, e.g., CH₃); (e.g., X″ is H or CH₃; e.g., X″ is H);    -   Y is C₆-C₁₀ aryl, which is optionally substituted with from 1-5        independently selected R^(b) (e.g., Y is unsubstituted phenyl);    -   R² is C₁-C₈ alkyl (e.g., C₁-C₆ alkyl, e.g., C₁-C₃ alkyl, e.g.,        CH₃, and    -   R¹² is C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g., ethoxy; e.g.,        containing branched alkyl, such a iso-propoxy) that is        optionally substituted with —NH₂, such as —OCH₂CH₃ or        —OCH₂CH₂NH₂; (see e.g., compounds 172-90 and 165-93).

In some embodiments of formula I-A:

-   -   X′ is S or NH;    -   X′″ is Y;    -   X″ is H or R^(a); R^(a) is C₁-C₈ alkyl (e.g., C₁-C₆ alkyl, e.g.,        C₁-C₃ alkyl, e.g., CH₃); (e.g., X″ is H or CH₃; e.g., X″ is H);    -   Y is C₆-C₁₀ aryl, which is optionally substituted with from 1-5        independently selected R^(b) (e.g., Y is unsubstituted phenyl);        and    -   R² is phenyl that is optionally substituted with from 1-4 R^(e)        (e.g., each occurrence of R^(e) is, independently, halo; cyano;        —C(O)(C₁-C₆ alkyl); C(O)OH; —C(O)O(C₁-C₆ alkyl); nitro; —NH₂;        —NH(C₁-C₆ alkyl), N(C₁-C₆ alkyl)₂, —NHC(O)(C₁-C₆ alkyl), C₁-C₆        alkoxy; C₁-C₆ haloalkoxy; C₁-C₆ thioalkoxy; C₁-C₆        thiohaloalkoxy; C₁-C₆ alkyl, C₃-C₆ cycloalkyl; and C₁-C₆        haloalkyl; or e.g., R² is unsubstituted phenyl).

In certain of these formula I-A embodiments:

-   -   X′ is S or NH;    -   X′″ is Y;    -   X″ is H or R^(a); R^(a) is C₁-C₈ alkyl (e.g., C₁-C₆ alkyl, e.g.,        C₁-C₃ alkyl, e.g., CH₃); (e.g., X″ is H or CH₃; e.g., X″ is H);    -   Y is C₆-C₁₀ aryl, which is optionally substituted with from 1-5        independently selected R^(b) (e.g., Y is unsubstituted phenyl);    -   R² is phenyl that is optionally substituted with from 1-4 R^(e)        (e.g., each occurrence of R^(e) is, independently, halo; cyano;        —C(O)(C₁-C₆ alkyl); C(O)OH; —C(O)O(C₁-C₆ alkyl); nitro; —NH₂;        —NH(C₁-C₆ alkyl), N(C₁-C₆ alkyl)₂, —NHC(O)(C₁-C₆ alkyl), C₁-C₆        alkoxy; C₁-C₆ haloalkoxy; C₁-C₆ thioalkoxy; C₁-C₆        thiohaloalkoxy; C₁-C₆ alkyl, C₃-C₆ cycloalkyl; and C₁-C₆        haloalkyl; or e.g., R² is unsubstituted phenyl); and    -   R¹² is    -   —C(O)OH; or    -   C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g., ethoxy; e.g., containing        branched alkyl, such a iso-propoxy) that is optionally        substituted with —NH₂, such as —OCH₂CH₃ or —OCH₂CH₂NH₂; or    -   heterocyclyl containing from 5-7 ring atoms, wherein from 1-2 of        the ring atoms of the heterocyclyl is independently selected        from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), NC(O)O(C₁-C₆        alkyl), O, and S; and each of which is optionally substituted        with from 1-3 independently selected C₁-C₄ alkyl groups, such as        an optionally substituted piperazinyl ring.

In certain of these formula I-A embodiments:

-   -   X′ is S or NH;    -   X′″ is Y;    -   X″ is H or R^(a); R^(a) is C₁-C₈ alkyl (e.g., C₁-C₆ alkyl, e.g.,        C₁-C₃ alkyl, e.g., CH₃); (e.g., X″ is H or CH₃; e.g., X″ is H);    -   Y is C₆-C₁₀ aryl, which is optionally substituted with from 1-5        independently selected R^(b) (e.g., Y is unsubstituted phenyl);    -   R² is phenyl that is optionally substituted with from 1-4 R^(e)        (e.g., each occurrence of R^(e) is, independently, halo; cyano;        —C(O)(C₁-C₆ alkyl); C(O)OH; —C(O)O(C₁-C₆ alkyl); nitro; —NH₂;        —NH(C₁-C₆ alkyl), N(C₁-C₆ alkyl)₂, —NHC(O)(C₁-C₆ alkyl), C₁-C₆        alkoxy; C₁-C₆ haloalkoxy; C₁-C₆ thioalkoxy; C₁-C₆        thiohaloalkoxy; C₁-C₆ alkyl, C₃-C₆ cycloalkyl; and C₁-C₆        haloalkyl; or e.g., R² is unsubstituted phenyl); and    -   R¹² is —C(O)OH (see, e.g., compound 165-74).

In some embodiments of formula I-A:

-   -   X′ is S or NH;    -   X′″ is Y;    -   X″ is H or R^(a); R^(a) is C₁-C₈ alkyl (e.g., C₁-C₆ alkyl, e.g.,        C₁-C₃ alkyl, e.g., CH₃); (e.g., X″ is H or CH₃; e.g., X″ is H);    -   Y is C₆-C₁₀ aryl, which is optionally substituted with from 1-5        independently selected R^(b) (e.g., Y is unsubstituted phenyl);        and    -   R² is heteroaryl containing from 5-6 (e.g., 5) ring atoms,        wherein from 1-4 of the ring atoms is independently selected        from N, NH, N(C₁-C₃ alkyl), NC(O)(C₁-C₆ alkyl), O, and S; and        wherein said heteroaryl is optionally substituted with from 1-3        R^(e), such as thienyl, furanyl, or thiazolyl; (e.g., each        occurrence of R^(e) is, independently, halo; cyano; —C(O)(C₁-C₆        alkyl); C(O)OH; —C(O)O(C₁-C₆ alkyl); nitro; —NH₂; —NH(C₁-C₆        alkyl), N(C₁-C₆ alkyl)₂, —NHC(O)(C₁-C₆ alkyl), C₁-C₆ alkoxy;        C₁-C₆ haloalkoxy; C₁-C₆ thioalkoxy; C₁-C₆ thiohaloalkoxy; C₁-C₆        alkyl, C₃-C₆ cycloalkyl; and C₁-C₆ haloalkyl; or e.g., R² is        unsubstituted heteroaryl, such as unsubstituted thienyl,        furanyl, or thiazolyl).

In certain of these formula I-A embodiments:

-   -   X′ is S or NH;    -   X′″ is Y;    -   X″ is H or R^(a); R^(a) is C₁-C₈ alkyl (e.g., C₁-C₆ alkyl, e.g.,        C₁-C₃ alkyl, e.g., CH₃); (e.g., X″ is H or CH₃; e.g., X″ is H);    -   Y is C₆-C₁₀ aryl, which is optionally substituted with from 1-5        independently selected R^(b) (e.g., Y is unsubstituted phenyl);    -   R² is heteroaryl containing from 5-6 (e.g., 5) ring atoms,        wherein from 1-4 of the ring atoms is independently selected        from N, NH, N(C₁-C₃ alkyl), NC(O)(C₁-C₆ alkyl), O, and S; and        wherein said heteroaryl is optionally substituted with from 1-3        R^(e), such as thienyl, furanyl, or thiazolyl; (e.g., each        occurrence of R^(e) is, independently, halo; cyano; —C(O)(C₁-C₆        alkyl); C(O)OH; —C(O)O(C₁-C₆ alkyl); nitro; —NH₂; —NH(C₁-C₆        alkyl), N(C₁-C₆ alkyl)₂, —NHC(O)(C₁-C₆ alkyl), C₁-C₆ alkoxy;        C₁-C₆ haloalkoxy; C₁-C₆ thioalkoxy; C₁-C₆ thiohaloalkoxy; C₁-C₆        alkyl, C₃-C₆ cycloalkyl; and C₁-C₆ haloalkyl; or e.g., R² is        unsubstituted heteroaryl, such as unsubstituted thienyl,        furanyl, or thiazolyl); and    -   R¹² is    -   —C(O)OH; or    -   C₁-C₆ alkoxy (e.g., C₂-C₆ alkoxy, e.g., ethoxy; e.g., containing        branched alkyl, such a iso-propoxy) that is optionally        substituted with —NH₂, such as —OCH₂CH₃ or —OCH₂CH₂NH₂; or    -   heterocyclyl containing from 5-7 ring atoms, wherein from 1-2 of        the ring atoms of the heterocyclyl is independently selected        from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), NC(O)O(C₁-C₆        alkyl), O, and S; and each of which is optionally substituted        with from 1-3 independently selected C₁-C₄ alkyl groups, such as        an optionally substituted piperazinyl ring.

In certain of these formula I-A embodiments:

-   -   X′ is S or NH;    -   X′″ is Y;    -   X″ is H or R^(a); R^(a) is C₁-C₈ alkyl (e.g., C₁-C₆ alkyl, e.g.,        C₁-C₃ alkyl, e.g., CH₃); (e.g., X″ is H or CH₃; e.g., X″ is H);    -   Y is C₆-C₁₀ aryl, which is optionally substituted with from 1-5        independently selected R″ (e.g., Y is unsubstituted phenyl);    -   R² is heteroaryl containing from 5-6 (e.g., 5) ring atoms,        wherein from 1-4 of the ring atoms is independently selected        from N, NH, N(C₁-C₃ alkyl), NC(O)(C₁-C₆ alkyl), O, and S; and        wherein said heteroaryl is optionally substituted with from 1-3        R^(e), such as thienyl, furanyl, or thiazolyl; (e.g., each        occurrence of R^(e) is, independently, halo; cyano; —C(O)(C₁-C₆        alkyl); C(O)OH; —C(O)O(C₁-C₆ alkyl); nitro; —NH₂; —NH(C₁-C₆        alkyl), N(C₁-C₆ alkyl)₂, —NHC(O)(C₁-C₆ alkyl), C₁-C₆ alkoxy;        C₁-C₆ haloalkoxy; C₁-C₆ thioalkoxy; C₁-C₆ thiohaloalkoxy; C₁-C₆        alkyl, C₃-C₆ cycloalkyl; and C₁-C₆ haloalkyl; or e.g., R² is        unsubstituted heteroaryl, such as unsubstituted thienyl,        furanyl, or thiazolyl); and    -   R¹² is —C(O)OH (see, e.g., compound 165-87).

In some embodiments:

A is N;

X contains 2 ring atoms independently selected from N, NH, N(C₁-C₃alkyl), O, and S, and one of the ring atoms is independently selectedfrom N, NH, and N(C₁-C₃ alkyl), and the other ring atom is independentlyselected from N, NH, N(C₁-C₃ alkyl), O, and S; and

R¹ is heteroaryl, which contains from 5-10 ring atoms, wherein from 1-4of the ring atoms is independently selected from N, NH, N(C₁-C₃ alkyl),O, and S; and wherein said heteroaryl ring is optionally substitutedwith from 1-3 independently selected R^(c).

In some embodiments, the compound has formula I-B:

wherein:

X′ is NH, O, or S; and

one of X″ and X′″ is Y, and the other of X″ and X′″ is H or R^(a).

Embodiments in which the compound has formula I-B can include one ormore of the following features.

X′ is S, X′″ is Y, and X″ is H or R^(a) (e.g., X″ is H).

X′ is NH, X′″ is Y, and X″ is H or R^(a) (e.g., X″ is H).

R¹ is thiazolyl.

Y is C₆-C₁₀ aryl, which is optionally substituted with from 1-5independently selected R^(b). R² is phenyl that is optionallysubstituted with from 1-4 R^(e). R² is unsubstituted phenyl.

Compound Forms and Salts

In some embodiments, the compounds described herein may contain one ormore asymmetric centers and thus occur as racemates and racemicmixtures, enantiomerically enriched mixtures, single enantiomers,individual diastereomers and diastereomeric mixtures (e.g., including(R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (+)(dextrorotatory) forms, (−) (levorotatory) forms, the racemic mixturesthereof, and other mixtures thereof). Additional asymmetric carbon atomsmay be present in a substituent, such as an alkyl group. All suchisomeric forms, as well as mixtures thereof, of these compounds areexpressly included in the present invention. The compounds describedherein may also or further contain linkages wherein bond rotation isrestricted about that particular linkage, e.g. restriction resultingfrom the presence of a ring or double bond (e.g., carbon-carbon bonds,carbon-nitrogen bonds such as amide bonds). Accordingly, all cis/transand E/Z isomers and rotational isomers are expressly included in thepresent invention. The compounds of this invention may also berepresented in multiple tautomeric forms; in such instances, theinvention expressly includes all tautomeric forms of the compoundsdescribed herein, even though only a single tautomeric form may berepresented. All such isomeric forms of such compounds are expresslyincluded in the present invention. Unless otherwise mentioned orindicated, the chemical designation of a compound encompasses themixture of all possible stereochemically isomeric forms of thatcompound.

Optical isomers can be obtained in pure form by standard proceduresknown to those skilled in the art, and include, but are not limited to,diastereomeric salt formation, kinetic resolution, and asymmetricsynthesis. See, for example, Jacques, et al., Enantiomers, Racemates andResolutions (Wiley Interscience, New York, 1981); Wilen, S. H., et al.,Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistry of CarbonCompounds (McGraw-Hill, N Y, 1962); Wilen, S. H. Tables of ResolvingAgents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of NotreDame Press, Notre Dame, Ind. 1972), each of which is incorporated hereinby reference in their entireties. It is also understood that thisinvention encompasses all possible regioisomers, and mixtures thereof,which can be obtained in pure form by standard separation proceduresknown to those skilled in the art, and include, but are not limited to,column chromatography, thin-layer chromatography, and high-performanceliquid chromatography.

The compounds of this invention include the compounds themselves, aswell as their salts and their prodrugs, if applicable. A salt, forexample, can be formed between an anion and a positively chargedsubstituent (e.g., amino) on a compound described herein. Suitableanions include chloride, bromide, iodide, sulfate, nitrate, phosphate,citrate, methanesulfonate, trifluoroacetate, and acetate. Likewise, asalt can also be formed between a cation and a negatively chargedsubstituent (e.g., carboxylate) on a compound described herein. Suitablecations include sodium ion, potassium ion, magnesium ion, calcium ion,and an ammonium cation such as tetramethylammonium ion. Examples ofprodrugs include C₁₋₆ alkyl esters of carboxylic acid groups, which,upon administration to a subject, are capable of providing activecompounds.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. As used herein, the term “pharmaceuticallyacceptable salt” refers to a salt formed by the addition of apharmaceutically acceptable acid or base to a compound disclosed herein.As used herein, the phrase “pharmaceutically acceptable” refers to asubstance that is acceptable for use in pharmaceutical applications froma toxicological perspective and does not adversely interact with theactive ingredient.

Examples of suitable acid salts include acetate, adipate, alginate,aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate,camphorate, camphorsulfonate, digluconate, dodecyl sulfate,ethanesulfonate, formate, fumarate, glucoheptanoate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate,pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,propionate, salicylate, succinate, sulfate, tartrate, thiocyanate,tosylate and undecanoate. Other acids, such as oxalic, while not inthemselves pharmaceutically acceptable, may be employed in thepreparation of salts useful as intermediates in obtaining the compoundsof the invention and their pharmaceutically acceptable acid additionsalts. Salts derived from appropriate bases include alkali metal (e.g.,sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄⁺ salts. This invention also envisions the quaternization of any basicnitrogen-containing groups of the compounds disclosed herein. Water oroil-soluble or dispersible products may be obtained by suchquaternization. Salt forms of the compounds of any of the formulaeherein can be amino acid salts of carboxy groups (e.g. L-arginine,-lysine, -histidine salts).

Lists of suitable salts are found in Remington's PharmaceuticalSciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418;Journal of Pharmaceutical Science, 66, 2 (1977); “Pharmaceutical Salts:Properties, Selection, and Use A Handbook; Wermuth, C. G. and Stahl, P.H. (eds.) Verlag Helvetica Chimica Acta, Zurich, 2002 [ISBN3-906390-26-8]; and Berge et al. (1977) “Pharmaceutical Salts”, J.Pharm. Sci. 66:1-19; each of which is incorporated herein by referencein its entirety.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the invention.

In addition to salt forms, the invention provides compounds which are ina prodrug form. Prodrugs of the compounds described herein are thosecompounds that undergo chemical changes under physiological conditionsto provide the compounds of the invention. Additionally, prodrugs can beconverted to the compounds of the invention by chemical or biochemicalmethods in an ex vivo environment. For example, prodrugs can be slowlyconverted to the compounds of the invention when placed in a transdermalpatch reservoir with a suitable enzyme or chemical reagent. Prodrugs areoften useful because, in some situations, they may be easier toadminister than the parent drug. They may, for instance, be morebioavailable by oral administration than the parent drug. The prodrugmay also have improved solubility in pharmacological compositions overthe parent drug. A wide variety of prodrug derivatives are known in theart, such as those that rely on hydrolytic cleavage or oxidativeactivation of the prodrug. An example, without limitation, of a prodrugwould be a compound of the invention which is administered as an ester(the “prodrug”), but then is metabolically hydrolyzed to the carboxylicacid, the active entity. In embodiments, the ester can be an alkyl ester(e.g., C₁-C₃ alkyl, e.g., CH₃ or CH₂CH₃; or C₃-C₆ alkyl, e.g., C₃-C₆branched alkyl, e.g., t-butyl, isopropyl, isobutyl). Additional examplesinclude peptidyl derivatives of a compound of the invention.

The invention also includes various hydrate and solvate forms of thecompounds (and salts thereof) described herein.

The compounds of the invention may also contain unnatural proportions ofatomic isotopes at one or more of the atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe invention, whether radioactive or not, are intended to beencompassed within the scope of the invention.

Synthesis of Compounds

The compounds described herein can be conveniently prepared inaccordance with the procedures outlined herein, from commerciallyavailable starting materials, compounds known in the literature, orreadily prepared intermediates, by employing standard synthetic methodsand procedures known to those skilled in the art. Standard syntheticmethods and procedures for the preparation of organic molecules andfunctional group transformations and manipulations can be readilyobtained from the relevant scientific literature or from standardtextbooks in the field. It will be appreciated that where typical orpreferred process conditions (i.e., reaction temperatures, times, moleratios of reactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvents used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures. Those skilled in the art of organic synthesiswill recognize that the nature and order of the synthetic stepspresented may be varied for the purpose of optimizing the formation ofthe compounds described herein.

Synthetic chemistry transformations (including protecting groupmethodologies) useful in synthesizing the compounds described herein areknown in the art and include, for example, those such as described in R.C. Larock, Comprehensive Organic Transformations, 2d.ed., Wiley-VCHPublishers (1999); P. G. M. Wuts and T. W. Greene, Protective Groups inOrganic Synthesis, 4th Ed., John Wiley and Sons (2007); L. Fieser and M.Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wileyand Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents forOrganic Synthesis, John Wiley and Sons (1995), and subsequent editionsthereof.

The processes described herein can be monitored according to anysuitable method known in the art. For example, product formation can bemonitored by spectroscopic means, such as nuclear magnetic resonancespectroscopy (e.g., ¹H or ¹³C), infrared spectroscopy (FT-IR),spectrophotometry (e.g., UV-visible), or mass spectrometry (MS), or bychromatography such as high performance liquid chromatograpy (HPLC) orthin layer chromatography (TLC).

Preparation of compounds can involve the protection and deprotection ofvarious chemical groups. The need for protection and deprotection, andthe selection of appropriate protecting groups can be readily determinedby one skilled in the art. The chemistry of protecting groups can befound, for example, in Greene, et al., Protective Groups in OrganicSynthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein byreference in its entirety.

The reactions of the processes described herein can be carried out insuitable solvents which can be readily selected by one of skill in theart of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,i.e., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of solvents. Depending on theparticular reaction step, suitable solvents for a particular reactionstep can be selected.

In some embodiments, the formula (I) compounds can be prepared using thereaction pathways and techniques as shown in FIGS. 2A-2I and 19A-19F. InFIG. 2B, compounds 161-87 and 153-96 can be prepared using this routeusing the corresponding thiazolyl and phenyl substituted reagents.

Pharmaceutical Compositions

The term “pharmaceutically acceptable carrier” refers to a carrier oradjuvant that may be administered to a subject (e.g., a patient),together with a compound of this invention, and which does not destroythe pharmacological activity thereof and is nontoxic when administeredin doses sufficient to deliver a therapeutic amount of the compound.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the compositions of this invention include, but are not limitedto, ion exchangers, alumina, aluminum stearate, lecithin,self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopherolpolyethyleneglycol 1000 succinate, surfactants used in pharmaceuticaldosage forms such as Tweens or other similar polymeric deliverymatrices, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts, orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium tri silicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat. Cyclodextrins such as α-, β-, andγ-cyclodextrin, or chemically modified derivatives such ashydroxyalkylcyclodextrins, including 2- and3-hydroxypropyl-β-cyclodextrins, or other solubilized derivatives mayalso be advantageously used to enhance delivery of compounds of theformulae described herein.

The compositions for administration can take the form of bulk liquidsolutions or suspensions, or bulk powders. More commonly, however, thecompositions are presented in unit dosage forms to facilitate accuratedosing. The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical excipient. Typical unit dosage forms includeprefilled, premeasured ampules or syringes of the liquid compositions orpills, tablets, capsules, losenges or the like in the case of solidcompositions. In such compositions, the compound is usually a minorcomponent (from about 0.1 to about 50% by weight or preferably fromabout 1 to about 40% by weight) with the remainder being variousvehicles or carriers and processing aids helpful for forming the desireddosing form.

The amount administered depends on the compound formulation, route ofadministration, etc. and is generally empirically determined in routinetrials, and variations will necessarily occur depending on the target,the host, and the route of administration, etc. Generally, the quantityof active compound in a unit dose of preparation may be varied oradjusted from about 1, 3, 10 or 30 to about 30, 100, 300 or 1000 mg,according to the particular application. In a particular embodiment,unit dosage forms are packaged in a multipack adapted for sequentialuse, such as blisterpack, comprising sheets of at least 6, 9 or 12 unitdosage forms. The actual dosage employed may be varied depending uponthe requirements of the patient and the severity of the condition beingtreated. Determination of the proper dosage for a particular situationis within the skill of the art. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small amounts until the optimumeffect under the circumstances is reached. For convenience, the totaldaily dosage may be divided and administered in portions during the dayif desired.

Use and Administration

This application features pyrazol-3-one compounds that activatepro-apoptotic BAX, making them therapeutically useful for treating(e.g., controlling, relieving, ameliorating, alleviating, or slowing theprogression of) or preventing (e.g., delaying the onset of or reducingthe risk of developing) diseases, disorders, and conditions associatedwith deregulated apoptosis of (e.g., diseased or damaged cells; e.g.,insufficient apoptosis of diseased or damaged cells; or lack of orreduced apoptosis of diseased or damaged cells). Examples of suchdiseases, disorders, and conditions include (but are not limited to)those associated with blockade(s) of cell death pathways (e.g.,over-expression of anti-apoptotic BCL-2 proteins), e.g.,hyperproliferative diseases, such as cancer. While not wishing to bebound by theory, it is believed that the compounds described hereininduce and increase apoptosis in target cells (e.g., pathogenic cellsincluding, but not limited to, cancer cells), thereby suppressing tumorgrowth and/or proliferation. It is further believed that increasingapoptosis in said target cells reestablishes the normal apoptoticcontrol that, during homeostasis, is associated with a regulated balancebetween pro- and anti-apoptotic protein functions.

The compounds and compositions described herein can, for example, beadministered orally, parenterally (e.g., subcutaneously,intracutaneously, intravenously, intramuscularly, intraarticularly,intraarterially, intrasynovially, intrasternally, intrathecally,intralesionally and by intracranial injection or infusion techniques),by inhalation spray, topically, rectally, nasally, buccally, vaginally,via an implanted reservoir, by injection, subdermally,intraperitoneally, transmucosally, or in an ophthalmic preparation, witha dosage ranging from about 0.01 mg/kg to about 1000 mg/kg, (e.g., fromabout 0.01 to about 100 mg/kg, from about 0.1 to about 100 mg/kg, fromabout 1 to about 100 mg/kg, from about 1 to about 10 mg/kg) every 4 to120 hours, or according to the requirements of the particular drug. Theinterrelationship of dosages for animals and humans (based on milligramsper meter squared of body surface) is described by Freireich et al.,Cancer Chemother. Rep. 50, 219 (1966). Body surface area may beapproximately determined from height and weight of the patient. See,e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 537(1970). In certain embodiments, the compositions are administered byoral administration or administration by injection. The methods hereincontemplate administration of an effective amount of compound orcompound composition to achieve the desired or stated effect. Typically,the pharmaceutical compositions of this invention will be administeredfrom about 1 to about 6 times per day or alternatively, as a continuousinfusion. Such administration can be used as a chronic or acute therapy.

Lower or higher doses than those recited above may be required. Specificdosage and treatment regimens for any particular patient will dependupon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the patient'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level. Patients may, however,require intermittent treatment on a long-term basis upon any recurrenceof disease symptoms.

In some embodiments, the compounds described herein can becoadministered with one or more other therapeutic agents. In certainembodiments, the additional agents may be administered separately, aspart of a multiple dose regimen, from the compounds of this invention(e.g., sequentially, e.g., on different overlapping schedules with theadministration of one or more compounds of formula (I) (including anysubgenera or specific compounds thereof)). In other embodiments, theseagents may be part of a single dosage form, mixed together with thecompounds of this invention in a single composition. In still anotherembodiment, these agents can be given as a separate dose that isadministered at about the same time that one or more compounds offormula (I) (including any subgenera or specific compounds thereof) areadministered (e.g., simultaneously with the administration of one ormore compounds of formula (I) (including any subgenera or specificcompounds thereof)). When the compositions of this invention include acombination of a compound of the formulae described herein and one ormore additional therapeutic or prophylactic agents, both the compoundand the additional agent can be present at dosage levels of betweenabout 1 to 100%, and more preferably between about 5 to 95% of thedosage normally administered in a monotherapy regimen.

The compositions of this invention may contain any conventionalnon-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles.In some cases, the pH of the formulation may be adjusted withpharmaceutically acceptable acids, bases or buffers to enhance thestability of the formulated compound or its delivery form.

The compositions may be in the form of a sterile injectable preparation,for example, as a sterile injectable aqueous or oleaginous suspension.This suspension may be formulated according to techniques known in theart using suitable dispersing or wetting agents (such as, for example,Tween 80) and suspending agents. The sterile injectable preparation mayalso be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example, as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that maybe employed are mannitol, water, Ringer's solution and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose, any blandfixed oil may be employed including synthetic mono- or diglycerides.Fatty acids, such as oleic acid and its glyceride derivatives are usefulin the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,or carboxymethyl cellulose or similar dispersing agents which arecommonly used in the formulation of pharmaceutically acceptable dosageforms such as emulsions and or suspensions. Other commonly usedsurfactants such as Tweens or Spans and/or other similar emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

The compositions of this invention may be orally administered in anyorally acceptable dosage form including, but not limited to, capsules,tablets, emulsions and aqueous suspensions, dispersions and solutions.In the case of tablets for oral use, carriers which are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions and/or emulsions are administered orally, the activeingredient may be suspended or dissolved in an oily phase is combinedwith emulsifying and/or suspending agents. If desired, certainsweetening and/or flavoring and/or coloring agents may be added.

The compositions of this invention may also be administered in the formof suppositories for rectal administration. These compositions can beprepared by mixing a compound of this invention with a suitablenon-irritating excipient which is solid at room temperature but liquidat the rectal temperature and therefore will melt in the rectum torelease the active components. Such materials include, but are notlimited to, cocoa butter, beeswax and polyethylene glycols.

Topical administration of the compositions of this invention is usefulwhen the desired treatment involves areas or organs readily accessibleby topical application. For application topically to the skin, thecomposition should be formulated with a suitable ointment containing theactive components suspended or dissolved in a carrier. Carriers fortopical administration of the compounds of this invention include, butare not limited to, mineral oil, liquid petroleum, white petroleum,propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifyingwax and water. Alternatively, the composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier with suitable emulsifying agents. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water. The compositions of thisinvention may also be topically applied to the lower intestinal tract byrectal suppository formulation or in a suitable enema formulation.

In some embodiments, topical administration of the compounds andcompositions described herein may be presented in the form of anaerosol, a semi-solid pharmaceutical composition, a powder, or asolution. By the term “a semi-solid composition” is meant an ointment,cream, salve, jelly, or other pharmaceutical composition ofsubstantially similar consistency suitable for application to the skin.Examples of semi-solid compositions are given in Chapter 17 of TheTheory and Practice of Industrial Pharmacy, Lachman, Lieberman andKanig, published by Lea and Febiger (1970) and in Remington'sPharmaceutical Sciences, 21st Edition (2005) published by MackPublishing Company, which is incorporated herein by reference in itsentirety.

Topically-transdermal patches are also included in this invention. Alsowithin the invention is a patch to deliver active chemotherapeuticcombinations herein. A patch includes a material layer (e.g., polymeric,cloth, gauze, bandage) and the compound of the formulae herein asdelineated herein. One side of the material layer can have a protectivelayer adhered to it to resist passage of the compounds or compositions.The patch can additionally include an adhesive to hold the patch inplace on a subject. An adhesive is a composition, including those ofeither natural or synthetic origin, that when contacted with the skin ofa subject, temporarily adheres to the skin. It can be water resistant.The adhesive can be placed on the patch to hold it in contact with theskin of the subject for an extended period of time. The adhesive can bemade of a tackiness, or adhesive strength, such that it holds the devicein place subject to incidental contact, however, upon an affirmative act(e.g., ripping, peeling, or other intentional removal) the adhesivegives way to the external pressure placed on the device or the adhesiveitself, and allows for breaking of the adhesion contact. The adhesivecan be pressure sensitive, that is, it can allow for positioning of theadhesive (and the device to be adhered to the skin) against the skin bythe application of pressure (e.g., pushing, rubbing,) on the adhesive ordevice.

The compositions of this invention may be administered by nasal aerosolor inhalation. Such compositions are prepared according to techniqueswell-known in the art of pharmaceutical formulation and may be preparedas solutions in saline, employing benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

A composition having the compound of the formulae herein and anadditional agent (e.g., a therapeutic agent) can be administered usingany of the routes of administration described herein. In someembodiments, a composition having the compound of the formulae hereinand an additional agent (e.g., a therapeutic agent) can be administeredusing an implantable device. Implantable devices and related technologyare known in the art and are useful as delivery systems where acontinuous, or timed-release delivery of compounds or compositionsdelineated herein is desired. Additionally, the implantable devicedelivery system is useful for targeting specific points of compound orcomposition delivery (e.g., localized sites, organs). Negrin et al.,Biomaterials, 22(6):563 (2001). Timed-release technology involvingalternate delivery methods can also be used in this invention. Forexample, timed-release formulations based on polymer technologies,sustained-release techniques and encapsulation techniques (e.g.,polymeric, liposomal) can also be used for delivery of the compounds andcompositions delineated herein.

EXAMPLES

The invention will be further described in the following examples. Itshould be understood that these examples are for illustrative purposesonly and are not to be construed as limiting this invention in anymanner.

Example 1 Identification of Small Molecules that Bind the BAX TriggerSite

We generated a diverse in silico compilation of 750,000 small moleculesfrom commercially available libraries and docked the database of3-dimensional molecules on average minimized BAX structures using Glide4.0^(19,20) in standard precision mode (SPVS) (FIG. 3A). The top-ranked20,000 hits based on the Glidescore function for each BAX structuralmodel were selected and re-docked to the BAX structures using extraprecision docking mode (XPVS). The top 1,000 hits from each dockingcalculation were visualized with the Glide pose viewer and analyzed fortheir interactions with key BAX binding site residues. A subset of 100molecules was selected for experimental analysis based on the presenceof favorable hydrogen bonds, hydrophobic contacts, and molecularproperties. Docking this compilation of putative BAX activator molecules(BAMs) demonstrates how the compounds blanket the surface of the BAXtrigger site (FIG. 3B).

To evaluate the capacity of candidate BAMs to bind BAX, we developed ascreening competitive fluorescence polarization assay (FPA) based on theinteraction between recombinant BAX and the fluoresceinated StabilizedAlpha-Helix of BCL-2 domain (SAHB) modeled after BIM BH3 (EC₅₀, 283 nM)(FIG. 4A). Small molecules were then benchmarked against thedisplacement of FITC-BTM SAHB by N-terminal acetylated BIM SAH (IC₅₀,314 nM) (FIG. 4B). Of the 78 molecules that lacked auto-fluorescence, 11molecules achieved >55% displacement of FITC-BIM SAHB at the 100 μMscreening dose (FIG. 4C). The ability of BAMs 1-11 to dose-responsivelycompete with FITC-BIM SAHB for BAX binding was then examined by FPA.BAM7 emerged as the most effective competitor, achieving an IC₅₀ of 3.3μM, which compared favorably with unlabeled BIM SAHB considering thatthe molecule is only one-sixth the size of the BIM BH3 α-helical peptide(FIG. 4D). We verified the identity of BAM7 by NMR, resynthesized it,and documented a similar IC₅₀ value for competition with FITC-BIM SAHB(FIG. 4D). The chemical structure of BAM7 (MW 405.5) is shown in FIG. 4Eand its ¹H-NMR spectra shown in FIG. 4F.

Example 2 BAM7 is Selective for the BH3-Binding Site on BAX

The BH3 binding pocket of anti-apoptotic targets shares topographicsimilarities with the BH3 trigger site on BAX, including a hydrophobicgroove that engages the hydrophobic face of BH3 helices and a perimeterof similarly oriented charged and polar residues that are complementaryto discrete residues of the hydrophilic BH3 interface. The two BH3binding sites differ in their geographic location, pocket depth, andfunctionality. Whereas BIM BH3 is compatible with both the BAX triggersite and anti-apoptotic pockets, we examined whether BAM7 was selectivefor BCL-2 family targets by competitive FPA. As demonstrated for BAX,direct FPA analyses documented high affinity interactions betweenFITC-BIM SAHB and the anti-apoptotic proteins BCL-XL (EC₅₀, 13.6 nM),MCL-1 (EC₅₀, 19.8 nM), and BFL-1/A1 (EC₅₀, 17.9 nM), which represent thestructural diversity of the pro-survival arm of the BCL-2 family (FIG.5A). Correspondingly, the N-terminal acetylated analogue of BIM SAHBeffectively competed with FITC-BIM SAHB for binding to BCL-XL (IC₅₀, 572nM), MCL-1 (IC₅₀, 136 nM), and BFL-1/A1 (IC₅₀, 603 nM) (FIGS. 5B-5D).These binding data highlight that BIM SAHB can readily engage thediversity of apoptotic targets. In striking contrast, BAM7 exhibitedlittle to no anti-apoptotic binding interactions even at 50 μM dosing,revealing a remarkable selectivity of BAM7 for BAX (FIGS. 4D and 5B-5D).

Example 3 Structural Analysis of the BAM7/BAX Interaction

To determine if BAM7 selectively competed with FITC-BIM SAHB for bindingto BAX through a direct trigger site interaction or an indirectallosteric effect, we performed NMR analysis of ¹⁵N-BAX upon BAM7titration. As observed for BIM SAHB_(A) ¹⁷, the addition of BAM7 up to a1:1 ratio induced significant backbone amide chemical shift changes inthose BAX residues concentrated in the region of the α1/α6 trigger site(FIG. 6A). These data are consistent with a direct interaction betweenBAM7 and BAX at the very surface employed by the BIM BH3 helix totrigger BAX activation.

We next performed molecular docking analysis to examine the predictedinteractions between BAM7 and the BAX trigger site. Interestingly, BAM7appears to insinuate itself along a crevice formed by residues locatedat (1) the junction between the α1-α2 loop's C-terminus and theN-terminus of α2, (2) the N-terminus of α1, and (3) the C-terminus of α6(FIG. 6B). This is an intriguing model of the complex from a functionalstandpoint, as engagement of this region by BIM SAHB is believed todisplace the α1-α2 loop and expose an epitope comprised of amino acids12-24, which are recognized by the 6A7 antibody only upon BAXactivation²¹. Indeed, the pyrazolone core of BAM7 sits at the base ofthe 6A7 activation epitope, with the carbonyl group engaged in hydrogenbonding interactions with K21, a key residue that participates incomplementary charge-charge interactions with E158 of the BIM BH3helix^(11,17). Whereas the ethoxyphenyl group abuts the confluence ofresidues at the α1-α2 loop's C-terminus and the N-termini of α1 and α2,a presumed hinge site for loop opening upon initiation of BAXactivation, the methyl and phenylthiazol R groups make hydrophobiccontact with that portion of the BIM BH3-binding groove formed byaliphatic residues of α1 and α6. Thus, docking analysis positions BAM7at a critical region of the BAX trigger site implicated inligand-induced α1-α2 loop displacement and resultant exposure of the 6A7activation epitope. This binding region is geographically andfunctionally distinct from the canonical BH3-binding site located at theC-terminal face of anti-apoptotic BCL-2 family proteins, and may accountfor the remarkable selectivity of BAM7 for BAX.

Example 4 BAM7 Activates BAX and BAX-Dependent Cell Death

In order to transform from an inactive cytosolic monomer into a toxicmitochondrial oligomer, BAX undergoes a major conformational change uponBH3 triggering. We recently demonstrated using correlative structuraland biochemical methods that these essential changes include “opening”of the α1/α2 loop, mobilization of the C-terminal α9 for mitochondrialtranslocation, and BAX BH3 exposure for propagating BAX activation¹¹. Todetermine if the selective binding interaction we documented for BAM7results in functional BAX activation, we performed a series ofstructural, biochemical, and cellular studies. First, we conducted anNMR analysis of ¹⁵N-BAX upon titration with higher concentrations ofBAM7 to examine secondary structural changes that ensue upon ligandbinding. We observed that increasing the ratio of BAM7/BAX from 1:1 to2:1 caused additional chemical shift changes in the α1-α2 loop, the BH3domain (α2), and in the C-terminal α9 helix, three discrete regions thatalso undergo allosteric changes in response to increased BIM SAHBexposure¹¹ (FIG. 7). To link these structural changes to the biochemicalconversion of BAX from monomer to oligomer, we performed solution-phaseBAX oligomerization assays in which BAX is exposed to increasingquantities of triggering ligand followed by monitoring of BAX species bysize-exclusion chromatography over time. Like BIM SAHB¹¹, BAM7 triggeredthe conversion of BAX from monomer to oligomer in a dose- andtime-responsive manner (FIG. 8A). To confirm that the SEC-baseddetection of BAM7-induced BAX oligomerization reflects functionalactivation of BAX for its release activity, we performed liposomalassays that explicitly evaluate the capacity of BAM7 to directly triggerBAX pore formation in the absence of other factors. Whereas treatmentwith BAX or BAM7 alone had no effect on the liposomes, the combinationof BAM7 and BAX yielded dose-responsive liposomal release of entrappedfluorophore (FIG. 8B). Thus, the direct interaction between BAM7 and BAXat the trigger site induces the characteristic structural changes thatyield functional BAX oligomerization.

Finally, we investigated whether this prototype BAX activator moleculethat directly, selectively, and functionally activates BAX in vitrocould induce BAX-dependent cell death. For these studies, we employedgenetically-defined mouse embryo fibroblasts (MEFs) that either expressonly BAX (Bak^(−/−)), only BAK (Bax^(−/−)) or neither death effector(Bax^(−/−)Bak^(−/−)). Thus, to undergo apoptosis, MEFs rely on BAX andBax^(−/−) MEFs rely on BAK, whereas Bax^(−/−)Bak^(−/−) MEFs areprofoundly resistant to apoptosis¹⁵. Strikingly, BAM7 dose-responsivelyimpaired the viability of Bak^(−/−) MEFs that exclusively express BAX,but had no effect on Bax^(−/−) MEFs that contain BAK but lack BAX (FIG.8C). Similar specificity of action in Bak^(−/−) MEFs was also observedfor formula (I) compounds (FIG. 9A-D). BAM7-treated Bak^(−/−) MEFslikewise exhibited characteristic microscopic features of apoptosis,including cellular shrinkage and membrane blebbing (FIG. 8E).Importantly, BAM7 did not affect the viability of Bax^(−/−) Bak^(−/−)MEFs, further confirming its specificity of action (FIG. 8C). Toevaluate the cytosolic vs. mitochondrial distribution of BAX in responseto BAM7 treatment, we transfected Bax^(−/−)Bak^(−/−) MEFs with EGFP-BAX,labeled mitochondria with MitoTracker, and then monitored BAXtranslocation by confocal fluorescence microscopy. We observed adose-responsive increase in BAX translocation as evidenced by conversionof the diffuse, cytosolic EGFP-BAX pattern to a mitochondrion-localizeddistribution (FIG. 8D).

Example 5 BAM7 Dose-Responsively Decreases Viability of DHL5 DiffuseLarge B-Cell Lymphoma Cells (DLBCL) and Synergizes with the BCL-2/BCL-XLInhibitor ABT-737

To assess the anti-cancer activity of BAM7, DHL5 DLBCL cells, which arerelatively resistant to ABT-737, were exposed to BAM7 anddose-responsive impairment of cell viability was observed at 24 hours,as assessed by CellTiterGlo (FIG. 10A). Adding a subcytotoxic dose ofthe selective BCL-2/BCL-XL inhibitor ABT-737 further sensitized thecells to BAM7 (FIG. 10A). Conversely, BAM7 sensitized DHL5 cells toABT-737 (FIG. 10B, 10C), which is otherwise less effective in DHL5 cellsdue to expression of anti-apoptotic proteins that lie outside itsbinding spectrum.

Thus, we find that BAM7 directly binds to the BAX trigger site andinitiates the characteristic structural changes that lead to functionalBAX activation. When applied to genetically-defined MEFs, BAM7 onlykills the cell line that contains BAX, inducing the morphologic featuresof apoptosis; in the context of imaging Bax^(−/−)Bak^(−/−) MEFs thatexpress EGFP-BAX, BAX translocation from cytosol to mitochondria, andattendant cellular shrinkage and membrane blebbing, is also observed.BAM7 impairs the viability of DHL5 lymphoma cells and can sensitize thecells to the BCL-2/BCL-XL inhibitor AB7-737. Taken together, thesestudies demonstrate the feasibility of targeting BAX with a selectivesmall molecule to trigger its pro-apoptotic activity.

Methods

In Silico Screening.

A diverse in silico library was generated from the followingcommercially available libraries downloaded from the ZINC database³⁴:ACB Blocks, Asinex, Chembridge, Maybridge, Microsource, NCI, Peakdale,and FDA-approved. The in silico library was filtered for drug-likefeatures, ADME properties, and appropriate functional groups withQikprop. Molecules were converted to 3D all-atom structures, generatinga maximum of 4 stereoisomers, ionization states for pH 7.0 and pH 2.0,and different tautomers with Ligprep. The database of in silico 3Dmolecules totaled approximately 750,000 compounds. BAX structures fordocking were prepared using an averaged BAX closed-loop structure and anaveraged BAX open-loop structure with GROMACS software. The twostructures were generated in suitable format for docking with Maestro.Docking was performed using Glide, with the small molecule database foreach BAX structure in standard precision mode (SPVS)¹⁹. The top 20,000hits based on Glidescore function were selected and redocked to the BAXstructures using extra precision docking mode (XPVS)²⁰. The top 1000hits from each docking calculation were visualized on the structure andthen analyzed for interactions with key BAX residues, leading toselection of 100 compounds for experimental screening.

BCL-2 Family Protein Production.

Recombinant and tagless full-length BAX, BCL-X_(L)ΔC, MCL-1ΔNΔC, andBFL-1/A1ΔC were expressed and purified as previously reported^(17,35).Transformed Escherichia coli BL21 (DE3) were cultured inampicillin-containing Luria Broth and protein expression was inducedwith 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The bacterialpellets were resuspended in buffer (250 mM NaCl, 20 mM Tris, completeprotease inhibitor tablet, pH 7.2), sonicated, and after centifugationat 45,000×g for 45 min, the supernatants were applied toglutathione-agarose columns (Sigma) for GST-BCL-XLAC, MCL-1ΔNΔC, andBFL-1/A1ΔC, or a chitin column (BioLabs) for Intein-BAX. On-beaddigestion of GST-tagged protein was accomplished by overnight incubationat room temperature in the presence of thrombin (75 units) in PBS (3mL), whereas the intein tag was cleaved from BAX by overnight incubationof the chitin beads at 4° C. with 50 mM DTT. BCL-X_(L)ΔC, MCL-1ΔNΔC, andBFL-1/A1ΔC were purified by size exclusion chromatography (SEC) using150 mM NaCl, 50 mM Tris, pH 7.4 buffer conditions, and full-lengthmonomeric BAX protein isolated by SEC using a Superdex-75 column (GEHealthcare) and 20 mM Hepes pH 7.2, 150 mM KCl buffer conditions.

Fluorescence Polarization Binding Assays.

Fluorescence polarization assays (FPA) were performed as previouslydescribed^(35,36). Briefly, direct binding curves were first generatedby incubating FITC-BIM SAHB (50 nM) with serial dilutions of full-lengthBAX, BCL-X_(L)ΔC, MCL-1ΔNΔC, or BFL-1/A1ΔC and fluorescence polarizationmeasured at 20 minutes on a SpectraMax M5 microplate reader (MolecularDevices). For competition assays, a serial dilution of small molecule oracetylated BIM SAHB (Ac-BIM SAHB) was combined with FITC-BIM SAHB (50nM), followed by the addition of recombinant protein at ˜EC₇₅concentration, as determined by the direct binding assay (BAX: 500 nM;BCL-X_(L)ΔC, MCL-1ΔNΔC, BFL-1/A1ΔC: 200 nM). Fluorescence polarizationwas measured at 20 minutes and IC₅₀ values calculated by nonlinearregression analysis of competitive binding curves using Prism software(Graphpad).

BAM7 Characterization by Mass Spectrometry and ¹H-NMR Spectroscopy.

4-(2-(2-ethoxyphenyl)hydrazono)-3-methyl-1-(4-phenylthiazol-2-yl)-1H-pyrazol-5(4H)-one. LC-MS: ES+406 (M+1). ¹H NMR (300MHz, DMSO-d6) d 7.96 (d, 2H, J=8.1 Hz), 7.86 (s, 1H), 7.75 (d, 1H, J=7.8Hz), 7.46 (t, 2H), 7.37-7.33 (m, 1H), 7.25-7.20 (m, 2H), 7.13-7.07 (m,1H), 4.29-4.22 (q, 2H), 2.36 (s, 3H), 1.48 (t, 3H).

NMR Samples and Spectroscopy.

Uniformly ¹⁵N-labeled full-length human BAX was generated as previouslydescribed^(17,37). Protein samples were prepared in 25 mM sodiumphosphate, 50 mM NaCl solution at pH 6.0 in 5% D₂O. BAM7 (10 mM stock)was titrated into a solution of 50 μM BAX to achieve the indicated molarratios. Correlation ¹H-¹⁵N HSQC spectra³⁸ were acquired at 25° C. on aBruker 800 MHz NMR spectrometer equipped with a cryogenic probe,processed using NMRPipe³⁹, and analyzed with NMRView⁴⁰. The weightedaverage chemical shift difference Δ at the indicated molar ratio wascalculated as √{square root over ({(ΔH)²+(ΔN/5)² }/2)} in p.p.m. Theabsence of a bar indicates no chemical shift difference, or the presenceof a proline or residue that is overlapped or not assigned. BAXcross-peak assignments were applied as previously reported³⁷. Thesignificance threshold for backbone amide chemical shift changes wascalculated based on the average chemical shift across all residues plusthe standard deviation, in accordance with standard methods⁴¹.

Structure Modeling.

Docked structures of BAX and BAM7 were generated using Glide 4.0^(19,20)(Schrodinger, 2006) and analyzed using PYMOL⁴².

BAX Oligomerization Assay.

BAM7 was added to a 200 □L solution (20 mM Hepes/KOH pH 7.2, 150 mM KCl,0.5% CHAPS) containing size exclusion chromatography (SEC)-purified,monomeric BAX at the indicated BAM7:BAX ratios. The mixtures and BAXmonomer alone were incubated at 30° C. for the indicated durations andthen subjected to analysis by SEC using an SD75 column and 20 mMHepes/KOH pH 7.2, 150 mM KCl running buffer. The monomeric andoligomeric fractions elute at ˜11.5-12.0 min and ˜6.5-7.5 min,respectively. Protein standards (GE Healthcare) were used to calibratethe molecular weights of gel filtration peaks. Replicates were performedusing at least two independent preparations of freshly SEC-purifiedmonomeric BAX protein.

Liposomal Release Assay.

Liposomes were prepared and release assays performed as previouslydescribed^(32,43). Liposomes were composed of the following molarpercentages of lipids (Avanti Polar Lipids): phosphatidylcholine, 48%;phosphatidylethanolamine, 28%; phosphatidylinositol, 10%; dioleoylphosphatidylserine, 10%; and tetraoleoyl cardiolipin, 4% and were loadedwith ANTS/DPX (Molecular Probe) upon extrusion. BAX (400 nM) wascombined with BAM7 (200 nM, 400 nM) in 96-well format (Corning) and thenliposomes were added (10 μL, from 50 μM total lipid stock) in assaybuffer (10 mM HEPES [pH 7], 200 mM KCl, 5 mM MgCl₂, and 0.2 mM EDTA) toa final volume of 100 Liposomal release was quantified based on theincrease in fluorescence that occurs when the ANTS fluorophore isseparated from the DPX quencher upon release from the liposomes into thesupernatant. Fluorescence (λ_(ex)=355 nm and λ_(em)=520 nM) was measuredfor 2 hours at 30° C. using a Tecan Infinite M1000 plate reader. Tomeasure maximal release, Triton X-100 was added to a final concentrationof 0.2% (v/v) after 2 h and fluorescence measured for an additional 10min. The percentage release of ANTS/DPX is calculated as percentagerelease=((F−F₀)/(F₁₀₀−F₀))×100, where F₀ and F₁₀₀ are baseline andmaximal fluorescence, respectively.

Cell Viability Assay.

Mouse embryonic fibroblasts (MEFs) cells were maintained in DMEM highglucose (Invitrogen) supplemented with 10% (v/v) FBS, 100 U/mLpenicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 50 mM HEPES, 0.1mM MEM non-essential amino acids and 50 μM β-mercaptoethanol MEFs(2.5×10³ cells/well) were seeded in 96-well opaque plates for 18-24hours and then incubated with serial dilutions of BAM7 or vehicle (0.15%DMSO) in DMEM at 37° C. in a final volume of 100 μl. DHL5 cells werecultured as described (Deng et al. Cancer Cell, 12, 171-185, 2007) andsubjected to vehicle, BAM7, ABT-737, and combinations thereof at theindicated doses. Cell viability was assayed at 24 hours by addition ofCellTiter-Glo reagent according to the manufacturer's protocol(Promega), and luminescence measured using a SpectraMax M5 microplatereader (Molecular Devices). Viability assays were performed in at leasttriplicate and the data normalized to vehicle-treated control wells.Leukemia cell viability and caspase 3/7 assays were performed asdescribed (Cohen et al, Chem Biol, 2012).

Light Microscopy.

MEFs (5,000 cells/well) were plated for 24 hours on glass bottom culturedishes (MatTek Corp., MA) and then incubated with BAM7 (15 μM) orvehicle (0.15% DMSO). Live cell imaging was performed using a TE2000-E2Nikon microscopy equipped with a temperature and CO₂-controlled chamberthat maintained an atmosphere of 3-5% humidified CO₂ at 37° C. AHamamatsu Orca ER digital CCD camera was used to capture images at 20×magnification for 24 hours at 20 min intervals. Acquisition, hardwarecontrol, and image analysis was performed using Nikon NIS-Elementssoftware.

BAX Translocation Assay.

MEFs were seeded on uncoated 24-well glass bottom plates at a density of2.5×10⁵ cells/well in 500 μL, of supplemented DMEM. After 6 hours, cellswere transfected using Lipofectamine™ 2000 (Invitrogen) according to themanufacturer's protocol, using 750 ng of plasmid DNA and 1.0 μL, ofLipofectamine™ per well. Plasmid DNA was generated by cloningfull-length BAX into the pEGFP-C3 plasmid (Clontech) using 5′ PstI and3′ XbaI restriction sites. After overnight transfection, cells weretreated with the indicated concentrations of BAM7 or vehicle (0.3% DMSO)in supplemented DMEM for 6 hours. Mitochondria were labeled withMitoTracker™ Red CMXRos (Invitrogen) according to the manufacturer'sprotocol using 20 nM probe in 500 μL supplemented, phenol red-free DMEMfor 15 minutes. The cells were then incubated in fresh media for anaddition 15 minutes prior to imaging. Confocal microscopy was performedusing a Yokogawa spinning disk confocal microscope (Yokogawa ElectricCorporation) equipped with a Nikon inverted Ti microscope. Solid statelasers set at 488 nm and 561 nm were used to visualize EGFP andMitoTracker® Red CMXRos, respectively. The plate temperature wasmaintained at 37° C. using an In Vivo environmental chamber (In VivoScientific). Images were collected using an Andor iXon DU-897 EM-CCDcamera (Andor Technology) and analyzed with ImageJ software (NIH).Percent EGFP-positive cells was determined by counting EGFP-positive andMitotracker-positive cells. Percent BAX translocation was calculated bydividing the number of cells containing mitochondrion-localized BAX bythe total number of EGFP-positive cells. Each treatment was performed inquadruplicate with >200 cells counted per well.

Example 6 Additional Biological Activity

FIGS. 15A-15G are graphs that demonstrate the anti-leukemic activity ofBAM7.

FIG. 16 is a graph that compares the anti-leukemic activity of BAM7 tocompounds 165-93, 165-60, and 172-90 (see FIG. 14). Other comparativedata is provided in the tables below:

FITC-BIM SAHB Compound Competition IC50 (μM) BAM7 6 172-19 2.7 172-90 2165-97 1.7 172-11 1.5

FIG. 17 is a graph that demonstrates the broad anti-leukemic activity ofcompound 172-90.

FIGS. 18A and 18B are graphs that demonstrate that compound 172-90overcomes the apoptotic resistance conferred by BCL-2 familyanti-apoptotic members BCL-XL and MCL-1; whereas the BCL-2/BCL-XLselective inhibitor ABT-737 induces cell death of the BCL-XL-dependentleukemia cell line, significant resistance to ABT-737 is manifest in theisogenic MCL-1 dependent leukemia cell line. In contrast, FIGS. 18C and18D are graphs that demonstrate that compound 172-90 inducesdose-responsive caspase 3/7 activity and cell death in both cell lines,overcoming formidable apoptotic resistance.

FITC-BIM SAHB Compound Competition IC50 (μM) BAM7 6 161-79 6.2 183-502.5 165-60 0.9

FITC-BIM SAHB Compound Competition IC50 (μM) BAM7 6 165-87 0.22 165-740.1

FITC-BIM SABH Compound Competition IC50 (μM) BAM7 6 161-87 2.4 153-96 2165-93 0.5

OTHER EMBODIMENTS

In some embodiments, compounds can have the 5,6 heteroring structurethat is present in compounds 165-90, 165-94, and 165-95 in FIG. 11. Inembodiments, the nitrogen in the 6-membered ring adjacent to thecarbonyl can be substituted with X-Y in which X and Y can be as definedanywhere herein; and the remaining positions can be substituted withsubstituents as defined in R² as defined anywhere herein.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-81. (canceled)
 82. A compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: A is N or CH; Xis heteroaryl, which contains 5 ring atoms and is optionally substitutedwith 1 R^(a), wherein 1 or 2 of the ring atoms is independently selectedfrom N, NH, N(C₁-C₃ alkyl), O, and S; Y is unsubstituted phenyl; R¹ isselected from the group consisting of: (i) heteroaryl, which containsfrom 5-10 ring atoms, wherein 1, 2, 3, or 4 of the ring atoms isindependently selected from N, NH, N(C₁-C₃ alkyl), O, and S; (ii)—C(O)-(heteroaryl), wherein the heteroaryl contains from 5-10 ringatoms, wherein 1, 2, 3, or 4 of the ring atoms is independently selectedfrom N, NH, N(C₁-C₃ alkyl), O, and S); and (iii) hydrogen; R² is C₁-C₈alkyl; and R^(a) is C₁-C₈ alkyl.
 83. The compound of claim 82, wherein Xis heteroaryl, which contains 5 ring atoms and is optionally substitutedwith 1 R^(a), wherein 1 or 2 of the ring atoms is independently selectedfrom N and S.
 84. The compound of claim 82, wherein X unsubstitutedthiazolyl or thiazolyl which is optionally substituted with 1 R^(a),wherein R^(a) is methyl.
 85. The compound of claim 82, wherein R¹ is a5-membered heteroaryl or a 10-membered heteroaryl, wherein 1 or 2 of thering atoms is independently selected from N, NH, and S.
 86. The compoundof claim 82, wherein R¹ is thiazolyl or indazolyl.
 87. The compound ofclaim 82, wherein R¹ is —C(O)-(heteroaryl), wherein the heteroarylcontains 5 ring atoms, wherein 1 or 2 of the ring atoms is independentlyselected from N and S.
 88. The compound of claim 82, wherein R¹ is—C(O)-(thiazolyl).
 89. The compound of claim 82, wherein R¹ is hydrogen.90. The compound of claim 82, wherein R² is methyl.
 91. The compound ofclaim 82, wherein the compound is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 92. A compound of Formula(I-A):

or a pharmaceutically acceptable salt thereof, wherein: X′ is S; X″ is Hor C₁-C₄ alkyl; X′″ is: (i) C₆-C₁₀ aryl, which is optionally substitutedwith 1, 2, 3, 4, or 5 independently selected R^(b) groups; or (ii)heteroaryl, which contains from 5-10 ring atoms, wherein 1, 2, 3, or 4of the ring atoms is independently selected from N, NH, N(C₁-C₃ alkyl),O, and S; and wherein said heteroaryl ring is optionally substitutedwith 1, 2, or 3 independently selected Rb groups; R² is: (i) C₁₋₄ alkyl;or (ii) unsubstituted heteroaryl containing from 5-6 ring atoms, wherein1, 2, 3, or 4 of the ring atoms is independently selected from N, NH,N(C₁-C₃ alkyl), NC(O)(C₁-C₆ alkyl), O, and S; R¹² is selected from thegroup consisting of H, methoxy, phenoxy, benzyloxy, C(═O)C₁₋₄ alkoxy,and —C(═O)—(C₁₋₄ alkoxy)-C(═O)-(phenyl); R¹³ is selected from the groupconsisting of H and C₂₋₄ alkoxy; R¹⁴ is selected from the groupconsisting of H, C₁₋₄ alkyl, C₁₋₄ alkoxy, NHC(═O)C₁₋₄ alkyl, andC(═O)C₁₋₄ alkoxy; wherein at least one of R¹², R¹³, and R¹⁴ is not H;and R^(b) is C₁₋₄ alkoxy; with the proviso that the compound of Formula(I-A) is not a compound selected from the group consisting of:


93. The compound of claim 92, wherein X″ is H or methyl.
 94. Thecompound of claim 92, wherein X′″ is phenyl, which is optionallysubstituted with 1 R^(b) group.
 95. The compound of claim 92, whereinX′″ is unsubstituted heteroaryl, which contains from 5-6 ring atoms,wherein 1 or 2 of the ring atoms is independently selected from N and S.96. The compound of claim 92, wherein R² is C₁₋₄ alkyl.
 97. The compoundof claim 92, wherein R² heteroaryl containing from 5-6 ring atoms,wherein 1 or 2 of the ring atoms is independently selected from N and S.98. The compound of claim 92, wherein R¹² is selected from the groupconsisting of H, methoxy, phenoxy, benzyloxy, —C(═O)OCH₂CH₃, and—C(═O)OCH₂C(═O)-phenyl.
 99. The compound of claim 92, wherein R¹³ is Hor ethoxy.
 100. The compound of claim 92, wherein R¹⁴ is selected fromthe group consisting of H, methyl, methoxy, ethoxy, —NHC(═O)CH₃, andC(═O)OCH₂CH₃.
 101. The compound of claim 92, wherein the compound isselected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 102. A pharmaceuticalcomposition comprising a compound of claim 82, or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier. 103.A pharmaceutical composition comprising a compound of claim 92, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 104. A method of treating cancer in a subject,comprising administering the compound of claim 82, or a pharmaceuticallyacceptable salt thereof, to a subject in need of cancer treatment in anamount effective to treat the cancer, wherein the cancer is leukemia.105. The method of claim 104, wherein the leukemia is selected from thegroup consisting of acute lymphoblastic leukemia (ALL) and acutemyelogenous leukemia (AML).
 106. A method of treating cancer in asubject, comprising administering the compound of claim 92, or apharmaceutically acceptable salt thereof, to a subject in need of cancertreatment in an amount effective to treat the cancer, wherein the canceris leukemia.
 107. The method of claim 106, wherein the leukemia isselected from the group consisting of acute lymphoblastic leukemia (ALL)and acute myelogenous leukemia (AML).
 108. A method of activating apro-apoptotic function of BAX in a cell or tissue containing BAX,comprising contacting the cell or tissue with a compound of claim 82, ora pharmaceutically acceptable salt thereof.
 109. A method of activatinga pro-apoptotic function of BAX in a cell or tissue containing BAX,comprising contacting the cell or tissue with a compound of claim 92, ora pharmaceutically acceptable salt thereof.